Generation Of Cabbage Centromere- Specific Histone Gene Cenh3 Mutant And GFP- Tailswap Transgenic Plants

Haploid plants contain only one single parental chromosome group.After doubling,DH line with pure genotype,consistent traits can be obtained quickly.Compared with the traditional breeding method,haploid breeding can greatly shorten the period,it is an efficient crop breeding pathway and of great significance for basic research such as gene function identification,cytology research and mutant screening.Haploid can be spontaneously induced or artificially induced,but the frequency of spontaneous production is extremely low,artificial induction costs are high and limited by the genotype of the plant,which largely restricts the development and application of haploids.In 2010,Ravi et al.developed a novel haploid-inducer based on centromeric protein-modified variants in the study of the Arabidopsis cenh3 mutant,replacing the original tail domain with the H3.3 histone tail domain and add a GFP protein tag at the terminus.GFP-tailswap gene was used to rescue the embryonic lethality of the cenh3 mutant.When the cenh3 mutant plant with GFP-tailswap gene crossed with other Arabidopsis thaliana,chromosomes derived from the GFP-tailswap plant all disappeared,forming a haploid containing only the other parental chromosome.This induction line efficiently produces male or female haploids in Arabidopsis.Due to the high conservation of CENH3 protein in eukaryotes,centromere-mediated induction of haploids has a wide range of adaptation possibilities for a variety of plants.Cabbage(Brassica oleracea)is an important vegetable crop of cruciferous family and its haploid breeding process marches slowly.It has great value in developing an efficient haploid inducer line for cabbage through the regulation of CENH3 gene expression.In this study,the CRISPR/Cas9 technology was used to simultaneously mutate the BoCENH3 gene and the regulatory gene BoSRK in SI reaction.Based on the construction of the GFP-tailswap gene expression vector,the transgenic plants were obtained through the transformation.The main results are as follows:1.Obtain cenh3 mutant background cabbage material.The premise of constructing a haploid inducer line based on CENH3 gene regulation is to down-regulate theexpression of endogenous CENH3 gene.In this study,the CRISPR/Cas technology was used to edit the CENH3 gene and the self-incompatible key gene SRK in the genome of Brassica oleracea.Double knockout vector pCas-tBoCENH3-ABCD/tBoSRK-ABCD with selection marker Bar gene was constructed.Agrobacterium tumefaciens mediated transformation of cabbage self-incompatible line 9025(SRK3 haplotype)was performed,we obtained 19 strains of pCas-tBoCENH3-ABCD/tBoSRK-ABCD transgenic plants(derived from a callus)and chose 12 plants for detection.Except plant No.6 was single gene knockout,others were all double gene knockout.The mutation frequency of BoCENH3 gene was 91.67%,the mutation frequency of BoSRK gene was100%,and the frequency of double mutation was 91.67%.Based on the results of monoclonal sequencing,all of the mutant plants were homologous knockout except that one was heterozygous mutation;comparing with the wild type strain,the knockout plant was short and the leaf margin was serrated during the seedling stage.Plant No.1 with large fragment missing has abnormal petals accompanied with thin and flat stamens.Pollen vigor and cytological observations showed that the pollen vigor of the knockout plants decreased and the pollen grains were malformed.The drug chamber was not cracked and no obvious pollen grains were formed;there was no significant difference in the development of ovules between the knockout plants and the wild type control,which preliminarily indicated that the female gametes of cenh3 mutant were not significantly affected;fluorescence microscopy observation of pollen in situ germination showed that when knockout plant was used as the female parent for flowering hybridization,pollen grains of the parental complementary plants were adsorbed on the surface of the stigma and formed a pollen tube through the stigma,indicating that the flowering stage hybridization is significantly different from the contol.It is preliminarily believed that the editing of the self-incompatibility gene SRK has an effect on the compatibility of the knockout plants;comparing with bud and flowering self-crossing process between knockout plants pCas-tBoCENH3-ABCD/tBoSRK-ABCD and wildtype control,the results showed that the elongation of the stigma during early stage of self-crossing was normal,but with the prolongation of time,the development gradually stopped and the phenomenon of dead embryo appeared,which was consistent with the results of the embryonic lethality of the cenh3 homozygous mutant.2.Obtainment of the transgenic cabbage pBoCENH3-GFPtailswap.The pBoCENH3-GFPtailswap complementary vector with selection marker Bar gene was constructed to rescue the embryonic lethality of cenh3.Agrobacterium tumefaciens-mediated transformation of the cabbage self-incompatible line 9025 was performed,we obtained seven pBoCENH3-GFPtailswap transgenic plants.PCR results showed that all plants contained complementary fragments;GFP fluorescence of the remaining 6 pollen grains and root tip tissues was detected.The results showed that the transgenic plants showed stronger GFP signals on the pollen wall comparing to wildtype 9025,but all showed no GFP signals in the nucleus.It may be related to the speculation that GFPtailswap protein cannot be loaded onto meiotic kinetochore;results of root tip tissue detection showed that the DAPI signal and GFP signal of transgenic plants pBoCENH3-GFPtailswap-5,6 can be co-localized in the nucleus,both transgenic plants' s GFP signals are significantly different from the control,indicating that GFP-tailswap protein can be loaded onto mitotic kinetochore.It showed that pBoCENH3-GFPtailswap-5,6 can be used for complement cenh3 mutant plants for obtaining a haploid inducer line.3.Knockout plants cross with complementary plants to obtain offspring.When the knockout plant pCas-tBoCENH3-ABCD/tBoSRK-ABCD was used as the female parent and the complementary plant pBoCENH3-GFPtailswap was used as the male parent for bud hybridization,some cross combinations can obtain seeds,but the seeding number is lower than the condition when the complementary plant was used as the female parent and the knockout plant was used as the male plant.It showed that the GFP-tailswap protein expressed in the ovule can improve the cenh3 mutation defect from the male gamete.