Study On The Expression Of Different Centromeric Histone CenH3 Variants In Cabbage

Cabbage（Brassica oleracea L.）is an annual or biennial plant in the Brassica genus of the cruciferous family.Cabbage has rich nutrition,strong adaptability and stress resistance,and is one of the important vegetable crops widely planted in many countries in the world.The breeding of cabbage crops relies on the utilization of heterosis.Due to its self-incompatibility in most of cabbage crops,the selection of its inbred lines requires artificial pollination for 7-8 generations of selfing or backcrossing,which is time-consuming and tedious jobs.Because haploids only contain a single parental chromosome,homozygous and consistent double haploids（DH）can be obtained by doubling colchicine,which greatly shortens the breeding period and saves labor costs.The centromere-specific histone CenH3（called CENP-A in humans）exists in the functional centromere of eukaryotes and plays an important role in the positioning of the centromere on the chromosome.Ravi and Chan found that the Arabidopsis CenH3protein was modified by replacing the H3 protein with H3.3 protein at the N-terminal tail of its conservative folding domain,and then adding green fluorescent protein marker GFP to obtain tailswap-GFP,the CenH3 variant It can complement the lethal phenotype of the cenh3/cenh3 mutant,and the complemented plant can induce25%-45%of haploid production when crossed as the female parent with the wild-type male parent.In addition,Sundaram kuppu et al carried out tiling identification and screening on mutants induced by EMS,and found that the change of single amino acid of CenH3 in Arabidopsis thaliana,such as the change of the 83rd amino acid of CenH3from glycine to glutamic acid（G83E）,or the change of the 83rd amino acid of CenH3from alanine to threonine（A155T）,can also induce haploid.In addition,2 amino acids deletion(G⁸⁶T⁸⁷)or 11 amino acids deletion(G⁸⁶TVALREIRHF⁹⁶)at conservative folding domainαN helix of the CenH3 also had the function of haploid induction.According to the successful haploid inducing results of a haploid inducer with CenH3 amino acid mutant in Arabidopsis thaliana,we constructed 6 amino acid mutants of CenH3 expression vectors:GA（G86E+A158T double mutation）,RA（R127C+A158T double mutation）and 2a（2 amino acids deletion inαN helix of CenH3）,11a（11 amino acids deletion inαN helix of CenH3）,CENm（G86E）and tailswap-GFP,The expression frames of each variant were linked with MYB,a gene related to anthocyanin synthesis,which was used for screening positive callus and seedling,and a seed specific expressed m Cherry gene,which can distinguish haploid and nonhaploid progenies at seed stage when the transgenic plants is taken as a haploid inducer.Agrobacterium tumefaciens mediated transformation method was used to transfer them into TO1000 cabbage.The main results are as follows:1.Generation and characterization of transgenic plants with amino acid variants of CenH3.The difficulty to transform different CenH3 variant genes into cabbage is different.Only 11a,2a,tailswap-GFP,CENm and GA variant genes generated purple callus after herbicide screening.And 18 rooting plants of the tailswap-GFP variant,11rooting plants of the 11a variant and 2 rooting plants of the GA variant were obtained.However,no purple callus was induced during the transformation of the RA variant genes.In the process of gene transformation of various CenH3 variants,spontaneous death of callus frequently occurred;root development was slow or even difficult to take root;the purple of the top leaves of the same pigment accumulation plant gradually faded and even naturally withered and died;the obtained transgenic plants were very easy to refine during seedling refinement.It dies due to loss of water,and neither hydroponics nor transplanting to the soil can survive.There was no significant difference in stomatal morphology between transgenic plants and the control transgenic plants with the same content accumulation of anthocyanin.Irregular GFP protein nuclear localization signals can be detected in the roots and leaves of tailswap-GFP transgenic plants.2.The expression analysis of the pigment synthesis related gene MYB and the exogenous and endogenous CenH3 genes.The expression level of MYB in red leaves was significantly higher than that in green leaves of tailswap-GFP variant transgenic plants.Compared with wild type,the expression levels of the 11a and tailswap-GFP variant genes in red and green leaf tissues are extremely low,however,the expression of endogenous CenH3 gene was significantly up-regulated in red and green leaves both of the 11a and tailswap-GFP transgenic plants.This result is similar to the result that the expression of corn Zm CENH3 is very low after introduction into wheat,but it induces the up-regulated expression of endogenous CenH3 in wheat.In addition,the overexpression of endogenous CenH3 in corn can cause death,which also is consistent with the frequent senescence and death of callus and transgenic plants found in this experiment.3.Gene expression involved into DNA damage repair and leaf senescence.The analysis of the expression of related genes in the red and green leaves of the 11a and tailswap-GFP variant transgenic plants showed that,in addition to NBS1,the DNA damage response genes ATM,ATR,SOG1,WEE1 as well as repair-related genes Ku70,Ku80,LIG4 and RAD51 all up-regulated their expression to varying degrees compared with the wild-type control,indicating that DNA damage and repair processes did occur in the transgenic plants.In the 11a,tailswap-GFP transgenic plants,transcription factors WRKY53 and ORE1,chlorophyll degradation PAO pathway related genes NYC1,SGR1,PPH,PAO as well as other senescence-related genes NAP,SAG12,SAG13,SAG113 were also involved to the senescence process of leaves at different degree,this result is consistent with the phenomenon of leaf senescence and death observed in the experiment.Therefore,we speculate that the expression of CenH3variant in the cabbage will cause the up-regulation of the endogenous CenH3 gene,which will affect cell mitosis,and the DNA damage causes cell cycle arrest,and incomplete DNA repair in time leads to cell apoptosis,which ultimately leads to plant death.