Expression Analysis Of 20 Candidate Genes Associated With Male Sterility In Hevea Brasiliensis And Functional Verification Of Some Genes In Transgenic Arabidopsis

Hevea brasiliensis is an important economic tree species in tropical regions.Male flower abortion breeding quality is an important resource for parent selection in hybrid breeding.The research group has screened several differentially expressed genes related to male sterility in rubber tree based on transcriptome sequencing data.In order to explore the function and regulation mechanism of these genes on male sterility of rubber tree,four rubber tree varieties,Reyan 93-114(Male sterility),Tianren 31-45(Male sterility),Reyan 7-33-97 and PR107,were used as materials in this study.20 candidate genes were selected for expression pattern analysis and functional verification.The expression patterns of candidate genes in different tissues and organs and in different developmental stages of male flowers were analyzed by q RT-PCR,and 10 important genes specifically expressed in male flowers were screened out.The expression of male flower-specific expression genes at different developmental stages was detected by m RNA-FISH to clarify the specific cell types of candidate genes expressed in male flowers.Eight genes specifically expressed in male flowers were selected to construct expression vectors,and transformed into Arabidopsis thaliana by inflorescence infection method.The correlation between candidate genes and male flower abortion was verified by morphological observation.This study aims to reveal the molecular regulation mechanism of male sterility in rubber tree.The main results are as follows:1.By analyzing the expression patterns of 20 candidate genes in shoot bark,tender leaves,latex,female flowers and male flowers,10 genes specifically expressed in male flowers were screened.DBR,EXLB1,ZAT5,MYB41 and b HLH91 were up-regulated in male flowers of sterile group,b HLH093,UND,At3g14460,BPC7 and At3g01820 were downregulated in male flowers of the sterile group.The expression levels of these genes were significantly higher in male flowers than in other tissues and organs and differed significantly between the fertile and sterile groups,and the abnormal expression in the sterile group may cause male flower abortion.2.By analyzing the expression levels of 20 candidate genes at different developmental stages of male flowers,the expression levels of DBR,ZAT5,MYB41,b HLH093,At3g14460,BPC7 and At3g01820 showed a low-high-low trend,with the highest relative expression at the tetrad period,and these genes may play a role in the tetrad period of male flowers;the expression levels of b HLH91 and UND showed a high-low-low trend,with the highest relative expression at the microspore mother cell stage,and these genes may play a role at the microspore mother cell stage in male flowers;the expression levels of EXLB1 showed a high-low-high trend in the fertile group and a low-high-low trend in the sterile group,with significant differences in the relative expression at the tetrad stage,and the gene may play a role in the tetrad period of male flowers.3.The expression characteristics of MYB41 and BPC7 in microspore mother cells,tetrads and mononuclear microspores of male flowers were analyzed by m RNA-FISH.It was detected that MYB41 was mainly expressed in anther epidermis and filaments,and BPC7 was mainly expressed in tapetum and tetrads.It is hypothesized that MYB41 has an effect on anther dehiscence and abnormal filament style development in sterile male flowers,and BPC7 has an effect on abnormal development of anther epidermis,filament style and tomentum in sterile male flowers,and abnormal expression of these genes in specific cell types leads to male flower abortion.4.The overexpression vectors of EXLB1,ZAT5,MYB41 and b HLH91 were cloned by gene cloning technology,and the knockout expression vectors of b HLH093,At3g14460,BPC7 and At3g01820 were constructed by CRISPR/Cas9 plant gene editing system.Four genes were successfully transformed into Arabidopsis thaliana by inflorescence infection method.Morphological observation showed that some transgenic Arabidopsis thaliana had abnormal floral organ development,and the length and number of pods were less than those of wild-type Arabidopsis thaliana.