Patent Application
20240409591
The present invention relates to the field of biotechnology and plant breeding, and in particular relates to a method using plant cell proliferation regulator gene to induce haploids, and a use thereof in plant breeding.
Compared with the traditional breeding methods, the haploid induction technology can obtain homozygous varieties within two generations, which can both shorten the breeding time and save unnecessary labor costs. There are two main source pathways for haploids, the first one comes from spontaneous formation in nature, and the second one comes from artificial methods to induce acquisition. Among them, the spontaneous formation of haploid ways in nature mainly include parthenogenesis, solitary male reproduction and sporophyte reproduction, but the occurrence frequency of these three pathways is very low (Zhang Zheng, 2007. Overview and thinking of research on haploid breeding. Shandong Agricultural Sciences, 5:122-125.DOI: 10.14083/j.issn.1001-4942.2007.05.025; Cui Ting, Li Yali, Qiao Linyi, Guo Huijuan, Dong Yanhui, Ren Yongkang, Tang Zhaohui, 2016. Wheat haploid breeding methods and its research progress. Shanxi Agricultural Sciences, 44 (1): 106-109.DOI: 10.3969/j.issn.1002-2481.2016.01.28), which cannot meet the needs of the breeding work. Breeders usually combine artificially induced haploids for assisted breeding.
There are many ways to induce haploids through artificial methods, and their induction rate is significantly higher than the frequency of natural occurrence. The methods for inducing parthenogenesis mainly include radiation induction, chemical induction, distant pollen stimulation induction, and induction using parthenogenesis induction lines (Xiang Zhiguo, Haiyan, Kang Minghui, Zhao Yong-ying, 2011. Haploid production way and its application in crop genetic breeding. Henan Agricultural Sciences, 40 (11): 17-21.DOI: 10.15933/j.cnki.1004-3268.2011.11.009). The methods for inducing solitary male reproduction mainly include: pollen in vitro culture, anther in vitro culture, and centromere mediated pathways (Li Ying, Wang Jia, Ji Lexiang, Li Hao, Ye Meixia, Guo Bin, Chen Zhong, An Xinmin, 2011, Research progress on plant haploid technology and its applications, Chinese Journal of Cell Biology, 33 (9): 1008-1014). The method of inducing sporophyte reproduction is called in vitro culture of female nuclei, which means in vitro culture of unpolluted organs such as ovaries or ovules to form new diploid plants. Haploid induction technology has been applied to breeding efforts in many crops, for example, rice, wheat, corn, cotton, barley, tobacco and rape. Its development is the most mature in maize, and has created a series of high frequency induction lines: WS14, EMK, Nongda Gaoyou 1 (NG1) and Ji Gaoyou 3 (JG3), among them, the induction rate of NG1 was 4% to 6%, the induction of JG3 was 10%. The application of these induced lines has greatly accelerated the process of maize breeding (Cai Zhuo, Xu Guoliang, Liu Xianghui, Dong Yalin, Dai Yuxian, Li Shuhua, 2007. The breeding of maize high frequency single reproductive induction line 3, Maize Science, 15(1):1-4.DOI: 10.13597/j.cnki.maize.science.2007.01.001).
In 2015, Conner et al. (Conner J A, Mookkan M, Huoh, Chae K, Ozias-Akins P, 2015. A parthenogenesis gene of apomict origin elicits embryo formation from unfertilized eggs in a sexual plant. PNAS, 112(36): 11205-11210.DOI: 10.1073/pnas.1505856112.) found that the PsASGR-BBML gene of African wolf tail grass was expressed in the ovary, developing seeds, roots and anthers before fertilization, and that the PsASGR-BBML transgene could promote the induction of pearl millet haploid embryos in sexual reproductive species. In 2017, Conner et al. (Conner J A, Podio M, Ozias-Akins P, 2017. Haploid embryo production in rice and maize induced by PsASGR-BBML transgenes. Plant Reprod, 30(1): 41-52.DOI: 10.1007/s00497-017-0298-x.) subsequent studies found that PsASGR-BBML transgenes induced the production of haploid embryos in two major monocot crops, maize and rice. In 2019, Khanday et al. (Khanday I, Skinner D, Yang B, Mercier R, Sundaresan V, 2019. A male-expressed rice embryogenic trigger redirected for asexual propagation through seeds. Nature, 565 (7737): 91-95.DOI: 10.1038/s41586-018-0785-8.) found that the expression of BBM1 in rice started from male gametes, activated the expression of the female genome, and then acted on the development process of the whole embryo. Ectopic expression of OsBBM1 in egg cells using AtDD45 promoter can induce parthenogenesis, with an induction rate of 5% to 10%, the induction rate of the T2transgenic material can reach 29%. The ectopic expression of BBM1 in eggs was combined with the MiMe (osd1/rec8/spoll-1) system, that is, the process of replacing meiosis in primitive germ cells with mitosis during parthenogenesis, and the whole genome was obtained.
In 2017, Kelliher, et al. (Kelliher T, Starr D, Richbourg L, Chintamanani S, Delzer B, Nuccio M L, Green J, Chen Z Y, Mccuiston J, Wang W L, Liebler T, Bullock P, Martin B, 2017. MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid Induction. Nature, 542 (7639): 105-109.DOI: 10.1038/nature20827.) found that the MTL gene is expressed in the cytoplasm of sperm, and the frameshift mutation could induce the production of haploid with an induction rate of 6.7%. In 2018, Yao et al. (Yao L, Zhang Y, Liu C X, Liu Y B, Wang Y L, Liang D W, Liu J T, Sahoo G, Kelliher T, 2018. OsMATL mutation induces haploid seed formation in indica rice. Nature Plants, 4(8): 530-533.DOI: 10.1038/s41477-018-0193-y.) in order to develop a haploid inducible gene in rice, OsMTL homologous to ZmMTL was found, and it was found that knockout of this gene can induce the production of haploid in rice, with an induction rate of 2%˜6%, which proves that MTL is also involved in mediating the formation of haploid in rice, and can be used to construct haploid inducible lines in rice. In 2019, Wang, et al. (Wang C, Liu Q, Shen Y, Hua Y F, Wang J J, Lin J R, Wu M G, Sun T T, Cheng Z K, Mercier R, Wang K J, 2019. Clonal seeds from hybrid rice by simultaneous genome engineering of meiosis and fertilization genes. Nature Biotechnology, 37 (3): 283-286.DOI: 10.1038/s41587-018-0003-0.) knockout out the OsMTL gene in CY84 of hybrid rice to obtain mtl haploid induction material of hybrid rice background with an induction rate of about 5% and seed set rate of about 10%. In addition, Fix (Fixation of hybrid) material was obtained by simultaneously knocking out four endogenous genes of REC8, PAIR1, OSD1 and MTL, completing the fixation of heterosis.
Although there are many methods to induce haploid production, there are few genes that can achieve haploid induction only by single gene regulation, and even fewer genes that can be applied to produce clonal seeds in rice apomicis. Currently, there are only two genes, OsBBM1 and OsMTL.
Through the analysis of expression profiles of rice unfertilized egg cells and embryos 5 days after fertilization, two genes with high expression were found, and two new genes with haploid induction ability were found in rice, named OsCPRO1 (CELL PROLIFERATION1) and OsCPRO2 (CELL PROLIFERATION2) respectively, the gene symbol of OsCPRO1 was LOC_Os03g47740, and the gene symbol of OsCPRO2 was LOC_Os12g43950. Moreover, these two haploid-inducible genes can be applied to apomixis in rice, realizing the fixation of heterosis and the formation of clonal seeds.
Two new genes with haploid induction ability, OsCPRO1 (LOC_Os03g47740) and OsCPRO2 (LOC_Os12g43950), were found in rice. This haploid induction material has high seed setting rate and can be applied to apomixis reproduction in rice.
Therefore, the invention provides a protein with haploid induction ability, including the POX superfamily domain and Homeobox domain, specifically, POX, the superfamily domain containing BELL domain and SKY domain: SKYLKAAQELLDEVVSV; preferably it is a OsCPRO1 or OsCPRO2 protein, amino acid sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 6, respectively, or their orthologous genes derived from the following species, optimally greater than 90%, 95%, more than 98% or 99%:rice (Oryza sativa), wart-grain rice (Oryza meyeriana var), maize (Zea mays), wheat (Triticum aestivum), durum wheat (Triticum turgidum subsp), wild emmer wheat (Triticum dicoccoides), urartu wheat (Triticum urartu), barley (Hordeum vulgare), section wheat (Aegilops tauschii), rye (Secale cereale), oats (Avena sativa), buckwheat (Fagopyrum esculentum), highland barley (Hordeum vulgare), Semen coicis (Coixlacryma-jobi), foxtail millet (Setariaitalica), fonio millet (Digitariaexilis), sorghum (Sorghum bicolor), proso millet (Panicum miliaceum), wild rice (Zizania latifolia), biotic wild rice (Zizania palustris), coracana (Eleusine coracana), sweet potato (Dioscorea esculenta), cassava (Manihot esculenta), potato (Solanum tuberosum), yam (Dioscoreapolystachya), groundnut (Arachis hypogaea), cranes peanut (Arachis duranensis), soybean (Glycine max), wild soybean (Glycine soja), adzuki bean (Vigna angularis), red bean (Vigna umbellata), lentil bean (Lens culinaris), mung bean (Vigna radiata), cowpea (Vigna unguiculata), kidney bean (Phaseolus vulgaris), broad bean (Vicia faba), pea (Pisum sativum), jack bean (Canavalia gladiata), pigeonpea (Cajanus cajan), white lupine (Lupinus albus), lather bean (Mucuna pruriens), narrow-leafed lupine (Lupinus angustifolius), hairy vine bean (Calopogoniummucunoides), chickpea (Cicer arietinum), sesame (Sesamum indicum), flax (Linumusitatissimum), rapeseed (Brassica napus), sunflower (Helianthus annuus), castor bean (Ricinus communis), red flower (Carthamus tinctorius), beet (Beta vulgaris), sugarcane (Saccharum officinarum), cotton (Gossypium spp), marijuana (Cannabis sativa), jiangnan rolling cypress (Selaginella moellendorffii), lemon eucalyptus (Corymbia citriodora), water green tree (Tetracentronsinense), mesquite tree (Prosopis alba), taxus chinensis (Taxus chinensis), olive (Olea europaea), African acacia (Abrusprecatorius), switchgrass (Panicum virgatum), red-vein maiguo (Rhamnellarubrinervis), American hickory (Carya illinoinensis), tea (Camellia sinensis), tobacco (Nicotiana tabacum), elephant grass (Pennisetum purpureum), hard-straight ryegrass (Lolium rigidum), nandi (Miscanthuslutarioriparius), canweed (Setariaviridis), cockweed (Pennisetum alopecuroides), Sudan grass (Sorghum sudanense), Tang pine grass (Thalictrum thalictroides), curved leaf thrush (Eragrostiscurvula), Brachypodium (Brachypodiumdistachyon), alfalfa (LotuscorniculatusL), Arabidopsis (Arabidopsis thaliana), Physcomitrella(Physcomitrium patens), hornmoss (Ceratodonpurpureus), cabbage (Brassica oleracea), mustard (Brassica juncea), Chinesegreen cabbage (Brassica rapa), cauliflower (Brassica oleracea), lettuce (Lactuca sativa), spinach (Spinacia oleracea), radish (Raphanus sativus), Chinese cabbage (Brassica rapa), zucchini (Cucurbita pepo), hemsley (Apium graveolens), leek (Allium tuberosum), carrot (Daucus carota), eggplant (Solanum melongena), tomato (Lycopersicon esculentum), cucumber (Cucumis sativus), wax gourd (Benincasahispida), pumpkin (Cucurbita moschata), loofah (Luffa cylindrica), vegetable melon (Cucumis melo), lotus vegetables (Potentilla anserina),day lily (Hemerocallis citrina), stem lettuce (Lactuca sativa), asparagus (Asparagus officinalis), chili pepper (Capsicum annuum), garlic (Allium sativum), spring onion (Allium fistulosum), Jerusalem artichoke (Helianthus tuberosus), Coriander (Coriandrum sativum), watermelon (Citrullus lanatus), pear (Pyrus spp), peach (Prunus persica), apricot (Armeniaca vulgaris), lee (Prunus salicina), date (Ziziphus jujuba),green plum (Vaticamangachapoi), apple (Malus pumila), sand fruit (Malus asiatica), cherry (Cerasus spp), walnut (Juglans regia), strawberry (Fragaria ananassa), grape (Vitis vinifera),pineapple pear (Ananas comosus), coptis chinensis (Coptis chinensis), Chinese milk vetch (Astragalus sinicus), ginseng (Panax ginseng), Angelica sinensis (Angelica sinensis), honeysuckle (Lonicera japonica), Ai (Artemisia argyi), peppermint (Mentha canadensis), orchid (Cymbidium ssp), lily (Lilium brownii), tulip (Tulipa gesneriana).
Correspondingly, the invention provides the protein encoding genes, which is either OsCPRO1 or OsCPRO2, where the gene symbol of OsCPRO1 is LOC_Os03g47740, the gene symbol of OsCPRO2 is LOC_Os12g43950; or BEL1-like homeodomain protein 6 derived from maize, Sequence ID: PWZ21223.1; or BEL1-like homeodomain protein 7 derived from wheat, Sequence ID: XP_044365192.1; or BEL1-like homeodomain protein 1 derived from soybean, Sequence ID: XP_003543416.1; or BEL1-like homeodomain protein 1 from groundpeanut, Sequence ID: XP_016170518.1; or at least 90%, 95% with the above genes, more preferably more than 98%, or 99% of the homologous genes derived from the same species, and still has the haploid induction ability.
More preferably, the nucleotide sequence of OsCPRO1 is as shown in SEQ ID NO: 1, or more than 90%, 95%, 98%, and still has haploid induction ability; the nucleotide sequence of OsCPRO2, as shown in SEQ ID NO: 4, is more preferably more than 98% and 99%, and still has haploid induction ability.
In addition, the CDS nucleotide sequence of OsCPRO1 is as shown in SEQ ID NO: 2, or from rice still has haploid induction ability with more than 98% or 99% identity, and still has haploid induction ability; the CDS nucleotide sequence of OsCPRO2 is as shown in SEQ ID NO: 5, or from rice still has more than 98% or 99% identity.
The invention provides an expression cassette, recombinant vector, recombinant cell or host cell containing the gene.
The invention also provides for the protein, or the application of the genes in inducing plant haploids.
The present invention further provides a method for inducing plant haploids using the gene, comprising the steps of transforming a plant with a recombinant expression vector containing the gene to obtain a positive transgenic plant; wherein the gene is controlled by the promoter of specific expression of the egg cell or, preferably, the plant includes a monocot and dicotyledon; Preferably, the promoter specifically expressed in egg cells is AtDD45 egg cells specific promoter, and more preferably, with the nucleotide sequence as described in SEQ ID NO: 7; the import is Agrobacterium-mediated or gene gun or gene editing.
More specifically, include the following steps: survey of wild-type plants and field phenotypic traits of T0transgenic-positive plants; Seeds of the T0transgenic positive material were replaced and germinated to obtain T1transtransgenic plants; using Indel markers or flow cell ploidy detection for T1generation of transgenic positive material for haploid detection to obtain haploid material. Further, including combining haploid induction material with the MiMe system to obtain apomictic material.
The haploid-induced genes found in the existing technologies are only OsBBM1 and OsMTL, which have been applied to the artificial creation of apomixis. Two new genes (OsCPRO1 and OsCPRO2) with haploid induction ability found by the present invention can also be applied to produce clonal seeds and have high application value.
The present invention is further explained in combination with the embodiments. These embodiments are used only to illustrate the invention and not to limit the scope of the invention.
Through the analysis of expression profiles of rice unfertilized egg cells and embryos 5 days after fertilization, two genes with high expression were found, and two new genes with haploid induction ability were found in rice, named OsCPRO1 (CELL PROLIFERATION1) and OsCPRO2 (CELL PROLIFERATION2) respectively, the gene symbol of OsCPRO1 was LOC 0s03g47740, and the gene symbol of OsCPRO2 was LOC_Os12g43950.
Homology alignment of amino acid sequences was performed using the NCBI database, including maize, wheat, soybean and groundnut. The results of amino acid sequence alignment of OsCPRO1 and OsCPRO2 homologous are as follows:
After verification, homologous genes in species include but are not limited to: rice (Oryza sativa), wart-grain rice (Oryza meyeriana var), maize (Zea mays), wheat (Triticum aestivum), durum wheat (Triticum turgidum subsp), wild emmer wheat (Triticum dicoccoides), urartu wheat (Triticum urartu), barley (Hordeum vulgare), section wheat (Aegilops tauschii), rye (Secale cereale), oats (Avena sativa), buckwheat (Fagopyrum esculentum), highland barley (Hordeum vulgare), semen coicis (Coixlacryma-jobi), foxtail millet (Setariaitalica), fonio millet (Digitariaexilis), sorghum (Sorghum bicolor), proso millet (Panicum miliaceum), wild rice (Zizania latifolia), biotic wild rice (Zizania palustris), coracana(Eleusine coracana), sweet potato (Dioscorea esculenta), cassava (Manihot esculenta), potato (Solanum tuberosum), yam (Dioscoreapolystachya), groundnut (Arachis hypogaea), cranes peanut (Arachis duranensis), soybean (Glycine max), wild soybean (Glycine soja), adzuki bean (Vigna angularis), red bean (Vigna umbellata), lentil bean (Lens culinaris), mung bean (Vigna radiata), cowpea (Vigna unguiculata), kidney bean (Phaseolus vulgaris), broad bean (Vicia faba), pea (Pisum sativum), jack bean (Canavalia gladiata),pigeonpea (Cajanus cajan), white lupine (Lupinus albus), lather bean (Mucuna pruriens), narrow-leafed lupine (Lupinus angustifolius), hairy vine bean (Calopogoniummucunoides), chickpea (Cicer arietinum), sesame (Sesamum indicum), flax (Linumusitatissimum), rapeseed (Brassica napus), sunflower (Helianthus annuus), castor bean (Ricinus communis), red flower (Carthamus tinctorius), beet (Beta vulgaris), sugarcane (Saccharum officinarum), cotton (Gossypium spp), marijuana (Cannabis sativa), jiangnan rolling cypress (Selaginella moellendorffii), lemon eucalyptus (Corymbia citriodora), water green tree (Tetracentronsinense), mesquite tree (Prosopis alba), taxus chinensis (Taxus chinensis), olive (Olea europaea), African acacia(Abrusprecatorius), switchgrass (Panicum virgatum), red-vein maiguo (Rhamnellarubrinervis), American hickory (Carya illinoinensis), tea (Camellia sinensis), tobacco (Nicotiana tabacum), elephant grass (Pennisetum purpureum), hard-straight ryegrass (Lolium rigidum), nandi (Miscanthuslutarioriparius), canweed (Setariaviridis), cockweed (Pennisetum alopecuroides), Sudan grass (Sorghum sudanense), Tang pine grass (Thalictrum thalictroides), curved leaf thrush (Eragrostiscurvula), Brachypodium (Brachypodiumdistachyon), alfalfa (LotuscorniculatusL), Arabidopsis (Arabidopsis thaliana), Physcomitrella(Physcomitrium patens), hornmoss (Ceratodonpurpureus), cabbage (Brassica oleracea), mustard (Brassica juncea), Chinesegreen cabbage (Brassica rapa), cauliflower (Brassica oleracea), lettuce (Lactuca sativa), spinach (Spinacia oleracea), radish (Raphanus sativus), Chinese cabbage (Brassica rapa), zucchini (Cucurbita pepo), hemsley (Apium graveolens), leek (Allium tuberosum), carrot (Daucus carota), eggplant (Solanum melongena), tomato (Lycopersicon esculentum), cucumber (Cucumis sativus), wax gourd (Benincasahispida), pumpkin (Cucurbita moschata), loofah (Luffa cylindrica), vegetable melon (Cucumis melo), lotus vegetables (Potentilla anserina), day lily (Hemerocallis citrina), stem lettuce (Lactuca sativa), asparagus (Asparagus officinalis), chili pepper (Capsicum annuum), garlic (Allium sativum), spring onion (Allium fistulosum), Jerusalem artichoke (Helianthus tuberosus), Coriander (Coriandrum sativum), watermelon (Citrullus lanatus), pear (Pyrus spp), peach (Prunus persica), apricot (Armeniaca vulgaris), lee (Prunus salicina), date (Ziziphus jujuba), green plum (Vaticamangachapoi), apple (Malus pumila), sand fruit (Malus asiatica), cherry (Cerasus spp), walnut (Juglans regia), strawberry (Fragaria ananassa), grape (Vitis vinifera), pineapple pear (Ananas comosus), coptis chinensis (Coptis chinensis), Chinese milk vetch (Astragalus sinicus), ginseng (Panax ginseng), Angelica sinensis (Angelica sinensis), honeysuckle (Lonicera japonica), Ai (Artemisia argyi), peppermint (Mentha canadensis), orchid (Cymbidium ssp), lily (Lilium brownii), tulip (Tulipa gesneriana).
The method of inducing haploid by OsCPRO1 and OsCPRO2 is summarized as follows (FIG. 9): {circle around (1)} selects the AtDD45 promoter specifically expressed in egg cells, and the nucleotide sequence, as shown in SEQ ID NO: 7, drives the CDS sequence or genome sequence of OsCPRO1 and OsCPRO2 genes respectively to construct ectopic expression vector; {circle around (2)} uses Agrobacterium-mediated method to obtain transgenic-positive material; {circle around (3)} investigates field phenotypic traits of wild-type CY84 and T0 transgenic-positive plants; {circle around (4)} Indel markers designed for haploid detection; {circle around (5)} statistics seed setting rate of T0trans-positive material and germination treatment to obtain T1generation of transgenic plants; {circle around (6)} using the Indel markers against T1generation of gene positive material; {circle around (7)} statistics haploid induction rate of T1transgenic plants; {circle around (8)} combines haploid induction material with the MiMe system to obtain apomixis material. The technical route profile is shown in
In this embodiment, the indica japonica hybrid rice Chunyou 84 (CY84) was selected as the genetic transformation material, CY84 is a hybrid breeding of the japonica sterile line Chunjiang 16A as the female parent and the indica restoring line C84 as the male parent, the hybrid rice has a longer growth period and is suitable for single season late rice cultivation. It has strong growth and large panicles with many grains. Due to its thick and robust stem, it has strong lodging resistance.
Select AtDD45 oocyte specific expression promoter to drive the CDS sequences of OsCPRO1 and OsCPRO2 genes, respectively, to complete the ectopic expression process.
(1) Arabidopsis leaf genomic DNA was used as a template to amplify the AtDD45 promoter sequence.
{circle around (1)} DNA extraction method: take about 2 cm of rice leaves into a 2 mL centrifuge tube with steel beads, 500 μL of CTAB extract was added; {circle around (2)} Transfer the centrifuge tube to the histiocyte shatter for shock grinding, 60 Hz/sec, 2 min; {circle around (3)} Place the grinding samples in a 65° C. water bath for 30 min, during this period, flip up and down for 10 min/time; {circle around (4)} After taking out the sample, centrifuge at 12000 rpm for 30 seconds, then add 300 μL of chloroform to the fume hood and mix upside down. {circle around (5)} i Centrifuge again at 12000 rpm for 8 minutes. After centrifugation, take 350 μL of the supernatant and place it into a 1.5 mL sterilized centrifuge tube. Add 700 μL of precooled anhydrous ethanol, shake well, and transfer to a −20° C. freezer for 15 minutes. {circle around (6)} After allowing the sample to stand still, centrifuge at 12000 rpm for 10 minutes. Discard the supernatant and air dry at room temperature. {circle around (7)} Add 100 μL of ddH2O for dissolution, gently shake, and store at 4° C. in the refrigerator for later use.
(2) RNA was extracted from CY84 and subsequently reverse transcribed into cDNA to provide a template for amplification of the CDS sequences of genes OsCPRO1 and OsCPRO2.
Methods for RNA extraction: {circle around (1)} Put 3-4 cm young rice panicles into a sterilized mortar pre cooled with liquid nitrogen, grind clockwise until they turn into a white green powder, and quickly transfer them to an EP tube without RNase containing 1 mL of Trizol. Vortex and shake for 1 minute to fully mix; {circle around (2)} Incubate at room temperature for 5 minutes, add 200 μL chloroform to the EP tube, vortex for 15 seconds, and let it stand on ice for 2 minutes; {circle around (3)} centrifuge at 12000 rpm at 4° C. for 15 minutes; {circle around (4)} Take 500 μL of supernatant, add 100 μL of chloroform, vortex for 15 seconds, and let it stand on ice for 2 minutes; {circle around (5)} i centrifuge at 12000 rpm at 4° C. for 15 minutes; {circle around (6)} Take 400 μL of the supernatant into a 1.5 mL EP tube without RNase, add 500 μL of isopropanol, invert and mix well; {circle around (7)} Let it stand on ice for 10 minutes, centrifuge at 12000 rpm at 4° C. for 10 minutes, and discard the supernatant; {circle around (8)} Add 1 mL of 75% ethanol, wash the precipitate with vortex shaking, centrifuge at 12000 rpm at 4° C. for 10 minutes, discard the supernatant and repeat the previous step; {circle around (10)} Retain the RNA precipitate at the bottom of the tube, remove the residual liquid, simply air dry, add 100 μL of RNase free water, dissolve with light tap, and test the concentration and purity. Store at −80° C. for later use.
Methods for RNA reverse transcription into cDNA: preparation of cDNA by HiFiScript cDNA Synthesis Kit (CW2569M). {circle around (1)} The reagent and RNA template provided in the kit were dissolved on ice for standby; {circle around (2)} mixed the reagent according to RNA reverse transcription system (Table 1), briefly centrifuged the liquid at the bottom of the tube; {circle around (3)} Subsequently, the reaction procedure was carried out, with 15 minutes at 42° C. followed by 5 minutes at 85° C.; {circle around (4)} Cool the cDNA on ice after the reaction for later use.
{circle around (3)} Design of the primers for amplification. The primers for the AtDD45 promoter sequence and the CDS sequences of the OsCPRO1 and OsCPRO2 genes are shown in Table 2, with lower case letters indicating adaptor primers.
{circle around (4)} The promoter sequences and the gene CDS sequences were amplified using the amplification primers. The amplification system is shown in Table 3, and the amplification procedure is shown in Table 4.
The pCAMBIA1300-ACTIN skeleton vector was digested with KpnI-HF and Bam HI-HF endonuclease, removing the original ACTIN promoter sequence, forming a gap at the sticky end, and retaining all other sequences including the original terminator. The restriction system is shown in Table 5.
2.3 the Recovered Amplified Fragments were Ligated to the Skeleton Vector
The ligation system is shown in Table 6. The mixing system is placed at 50° C. for 15 min. After ligation, the product is placed on ice.
Method of transforming the ligation products: {circle around (1)} Remove the competent cells stored in the −80° C. refrigerator (provided by the laboratory) and put them on ice for dissolution; {circle around (2)} When the competent cell (50 μL˜100 μL) reaches the molten state, add the ligation products immediately; {circle around (3)} Set still on the ice for 30 min; {circle around (4)} 42° C. water bath heat shock 90 sec; {circle around (5)} i 500 μL of liquid LB medium was added; {circle around (6)} 37° C. shaker culture for 1 h; {circle around (7)} 4000 rpm, 3 min, slowly centrifuge and apply onto LB solid culture medium with Kan resistance; {circle around (8)} Incubate overnight at 37° C. and perform colony PCR detection after the plaque grows. Select a single colony and culture it in Kan resistant liquid medium on a constant temperature shaker at 200 rpm and 37° C. for 14 hours. Extract plasmids and perform Sanger sequencing.
The ectopic expression vector was transferred into Agrobacterium tumefaciens strain EHA105 by electroshock and transferred into CY84 in hybrid rice using Agrobacterium-mediated method.
Specific method of transformation: {circle around (1)} Sterilize the embryos of hybrid rice CY84 seeds; {circle around (2)} Inoculate into the culture medium for inducing callus tissue; {circle around (3)} After one week of cultivation, select embryogenic callus tissue that is vigorous in growth, light yellow in color, and relatively loose as the receptor for transformation; {circle around (4)} Infect rice callus tissue with EHA105 strains containing pC1300-AtDD45P-OsCPRO1 and pC1300-AtDD45P-OsCPRO2 plasmids respectively; {circle around (5)} Incubate in the dark at 25° C. for 3 days; {circle around (6)} Screening resistant callus tissues and transgenic plants using a selective culture medium containing 50 mg/L hygromycin; {circle around (7)} Select the transgenic plants for normal growth.
The transgenic plants are planted in transgenic experimental fields in summer and in greenhouses in winter. The average temperature during the day in the greenhouse is 34° C., the average temperature at night is 25° C., there is a 12 hour/12 hour dark cycle, and the relative humidity is 75%.
Primers designed for the AtDD45 promoter sequence and the gene CDS sequence (spanning the intron region) were also used as detection primers for transgenic positive plants. The specific primer information is shown in Table 7.
Field phenotype survey includes: plant type, ear type and seed set rate.
6.1 Mutant strain of plant type (
6.2 Statistics of seed set rate: 3 rice ears per plant were selected for statistics, and the calculation formula: seed set rate=(real grain number/total grain number) 100%.
According to the whole genome sequence of hybrid rice CY84 and its two parents (16A and C84), a pair of Indel markers were designed on each chromosome of rice, a total of 12 pairs of primer markers, primer information is shown in Table 10, genotyping is shown in
The haploid or diploid plants screened by Indel markers were further confirmed for cell ploidy using flow cytometry (
The method of flow cytometry ploidy detection: {circle around (1)} Cut about 3 cm of fresh young leaves and place them in sterilized glass petri dishes. Place the petri dishes in a tray filled with ice, and perform the following on ice; {circle around (2)} Add 1 mL of plant lysis buffer (LB01) to the petri dish, cut the leaves with a blade, and immerse the tissue cells of the leaves into the lysis buffer; {circle around (3)} Extract the lysis solution from the petri dish, filter it with 50 μm nylon mesh into a 1.5 mL sterile centrifuge tube, and store it on ice. Centrifuge at 1200 rpm at 4° C. for 5 minutes; {circle around (5)} i Discard the supernatant and add pre cooled 450 μL of LB01, 25 μL of PI (1 mg/mL), and 25 μL of RNaseA (1 mg/mL); {circle around (6)} Staining on ice in the dark for 10 minutes; {circle around (7)} Detecting with BD Accuri C6 machine.
Select the parents 16A and C84 of hybrid rice CY84, and CY84, as well as the leaves of haploid plants, and extract DNA for whole genome sequencing. The whole genome sequencing results show that there are many different homozygous genotypes between 16A and C84; The genotype of CY84 at these loci is a heterozygous genotype that includes both the 16A genotype and the C84 genotype; while the haploid plant at these loci with homozygous genotypes of 16A or C84.
The AtDD45 egg specific expression promoter was selected to drive the genomic sequence of OsCPRO1 and OsCPRO2 genes respectively to complete the ectopic expression process.
Primers were designed on the AtDD45 promoter sequence for detection of transgenic-positive plants. See primer information in Table 7 in embodiment 1, Table 8 in embodiment 1 for detection system, and Table 9 in embodiment 1 for amplification procedures.
1. Experimental materials: wild Arabidopsis Columbia (Col-0) ecotype was used as the experimental materials.
2. Ectopic expression vector construction
The egg specific expression promoter of AtDD45 was selected to drive the CDS sequences of AtCPRO1 and AtCPRO2 genes respectively to complete the ectopic expression process.
The ectopic expression vector was transferred into Agrobacterium tumefaciens strain GV 310 by electroshock and transferred into Col-0 ecotype wild A. thaliana using Agrobacterium-mediated dipping method.
Specific method of transformation: {circle around (1)} Immerse the inflorescence of T0 generation Arabidopsis in a suspension of Agrobacterium tumefaciens strain GV310 containing the target transformation plasmid at a certain concentration; {circle around (2)} Cultivate under certain conditions; {circle around (3)} Collect seeds of T0 generation Arabidopsis after maturity; {circle around (4)} Place these seeds on a culture medium containing specific antibiotics for growth screening and obtain positive plants.
The transgenic material was grown in a light incubator. Light treatment for 15 hours, with an average temperature of 24° C.; dark treatment for 9 hours, with an average temperature of 22° C. The average humidity of the soil is 75%, and the nutrient soil: vermiculite=1:1. Water the nutrient solution every 3-4 days and immerse it from the bottom of the plate.
Design primers on the CDS sequence of genes, spanning intron regions, and use this primer as a detection primer for transgenic positive plants. The detection system can be found in Table 8 of embodiment 1, and the amplification program can be found in Table 9 of embodiment 1.
Phenotypic trait surveys included plant type and seed set rate.
6.1 Investigation of the plant type of the T2 generation (
6.2 Statistics of Seed Setting Rate: Collect mature but not yet cracked wild type and mutant Arabidopsis longhorned fruits, put them into a decolorization solution (ethanol: acetic acid=3:1) for transparent decolorization, and when the seeds in the pods can be clearly seen, take them out and place them under a microscope to calculate the seed setting rate.
Confirm the cell ploidy of plants using flow cytometry technology, and refer to embodiment 1 for the method of flow cytometry ploidy detection.
1. Experimental materials: see embodiment 1.
2. Construct an ectopic expression vector: see embodiment 1.
MiMe material was constructed by using the CRISPR-Cas 9 multigene knockout system to simultaneously knockout OsOSD1, OsPAIR1 and OsREC8 rice endogenous genes, with the main steps as follows (see CN201510485573.2 for specific operational details):
(1) Design of target sequence: search for NNNNNNNNNNNNNNNNNNN (NGG) specific sequence at the target position of the gene, with GC content between 35% and 75%. The following four sites were selected as sites for knockout OsOSD1, OsPAIR1 and OsREC8 by CRISPR-Cas 9 gene editing system (underlined PAM sequence):
(2) Primer design: add four bases of GGCA before the forward target sequence and four bases of AAAC before the reverse target sequence, respectively named g++ and g−−, performed the primer synthesis.
(1) The Aar I enzyme was used to digest the intermediate vector SK-gRNA to form the sticky ends, and the digestion system is shown in Table 11.
(2) Mix 20 μL of 100 μM g++ and g−− primers in equal proportions, and incubate at 100° C. for 5 minutes to form sticky ends after denaturation annealing.
(3) Ligating via T4 DNA ligase and ligate at room temperature for 1 hour. The ligation system is shown in Table 12.
(4) Transformation ligation products: see embodiment 1.
Utilizing the properties of BamHI and BglII being homologous enzymes, the polymerization of three SK-gRNA intermediate vectors was completed using T4 DNA ligase. As the final vector, KpnI and BamHI were used for digestion, while KpnI and BglII were used for digestion of the fragments provided. The ligation between SK-gRNAs was completed using two pairs of homologous enzymes NheI/XbaI and SalI/XhoI. The system with multiple intermediate vectors ligated is shown in Table 13.
The ectopic expression vector and the multigene knockout vector were combined to construct a binary fusion expression vector. Among them, the ectopic expression vector provided the desired fragment, and the multigene knockout vector was used as the backbone vector for enzyme digestion.
The whole fragment of the ectopic expression process was amplified from the ectopic expression vector, including three parts: AtDD45 promoter fragment, OsCPRO1 and OsCPRO2 gene CDS sequence and terminator sequence. The amplification primers are shown in Table 14 and lower letters indicate the adaptor primers.
Using PmeI endonuclease, the pC1300-Cas9-gRNAOSD1-gRNAPAIR1-gRNAREC8 multi gene knockout vector was digested. The digestion system is shown in Table 15.
The ligation system is shown in Table 16. The mixing system is placed at 50° C. for 15 min, and the product is placed on ice after the ligation.
4.4 Transformation of the ligation products: See embodiment 1.
5. Get transgenic plants: see embodiment 1.
6. Plant transgenic plants: see embodiment 1.
7. Transgenic plants detection
7.1 Positive detection of transgenic plants: see embodiment 1.
The materials with successful binary fusion expression were selected, that is, when the AtDD45 promoter drives the successful expression of the CDS sequence of the OsCPRO1/OsCPRO2 gene, OsOSD1, OsPAIR1 and OsREC8 were successfully knocked out (MiMe). The seeds of this material are germinated and planted (first filial generation).
We screened the cell ploidy by flow cytometry to select the first filial generation plants with consistent cell ploidy and the parent plant as apomixis material.
10. Analysis of whole-genome sequencing: see embodiment 1.
1. Experimental materials: see embodiment 1.
2. Construction of an ectopic expression vector: see embodiment 2.
3. Construction of multigene knockout vectors: see embodiment 4.
4. Construct a binary fusion expression vector
The ectopic expression vector and the multigene knockout vector were combined to construct a binary fusion expression vector. Among them, the ectopic expression vector provided the desired fragment, and the multigene knockout vector was used as the backbone vector for enzyme digestion.
The genomic sequence of the AtDD45 egg-specific expression promoter driving the OsCPRO1 and OsCPRO2 genes, completing the whole fragment of the ectopic expression process, was amplified from the ectopic expression vector, containing three parts: the AtDD45 promoter fragment, the genomic sequence of the OsCPRO1 and OsCPRO2 genes, and the terminator sequence. The amplification primers are shown in Table 14 in embodiment 3.
4.2 To digest multiple gene knockout vector: see embodiment 4.
4.3 Attach the amplified fragments to the multigene knockout vector: see embodiment 4.
4.4 Transformation of the ligation products: see embodiment 1.
5. Get transgenic plants: see embodiment 1.
6. Plant transgenic plants: see embodiment 1.
7. Transgenic plants detection
7.1 Positive detection of transgenic plants: see embodiment 2.
7.2 Detection of target site knockout: see embodiment 4.
8. Investigate field phenotypes: see embodiment 1.
9. Flow cytometric ploidy detection: see embodiment 4.
10. Analysis of whole-genome sequencing: see embodiment 1.
For embodiment 1 and 2 in Example 2, investigation of field phenotypes of T0transgenic positive plants revealed that compared with the wild-type CY84, the transgene positive material had no significant difference in vegetative growth, and the seed set rate was reduced to different degrees, including 80-100%, 50-80%, 50-30-50%, 10-30%, and 0-10%. The plant type was summarized in
For embodiment 1 and 2 in Example 2, 12 pairs of Indel markers used to screen haploids and double haploids showed obvious polymorphism and could be used for genotype identification of transgenic materials, as shown in
For screening the haploid material or double haploid material in Example 2, the test results of the CDS sequence of OsCPRO2 in Example 1:12 Indel marker sites showed single band genotype (
For the haploid and double haploid materials selected from embodiment 1 and 2 in Example 2, taking the detection results of AtDD45 oocyte specific expression promoter driving OsCPRO2 to achieve ectopic expression in embodiment 1 as an example, compared with the wild-type CY84, haploid materials were significantly affected in terms of nutritional and reproductive growth: plant height decreased, spike length shortened, glume reduced, and fertility completely lost (
For the T1 generation transgenic positive materials of embodiment 1, 2, and 3 in Example 2, it was found that the materials with haploid induction ability had a variation range of 18.98±2.14%˜-50.88±0.74% in seed setting rate and 0.44%˜7.14% in induction rate. The specific statistical information is shown in Table 18. Testing was conducted on the T1 generation transgenic positive materials of embodiment 4 and 5 in Example 2, and it was found that the fusion free reproductive materials produced by ectopic expression of OsCPRO1 and OsCPRO2 genes in oocytes combined with the MiMe system ranged in proportion to cloned seeds from 0.95% to 7.92%. The specific statistical information is shown in Table 19.
Arabidopsis
thaliana
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210675450.5 | Jun 2020 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2023/098723 | 6/6/2023 | WO |
