The Expression Of Key Genes Of Male Sterility Associated With Anther Development And Its FISH Analysis With HbAPXs Gene Family In Hevea Brasiliensis

Rubber is also known as one of the four industrial raw materials,it is a very important material strategic resources.Hevea brasiliensis(commonly known as rubber trees)is currently the only source of natural rubber,and it is also an important cash crop in tropical and subtropical regions of China.The male flower abortion germplasm of rubber tree is an important resource in the cross breeding of rubber tree,however,the mechanism of male flower abortion in rubber trees is not clear.The germplasms of male flower abortion in rubber trees are important resources for parents selection in rubber tree cross breeding.In this study,male sterility related candidate genes were selected by our research group,16 candidate genes associated with anther development were selected,thermal research of sterile varieties 93-114 and Tian Ren 31-45,a total of 4 varieties of rubber trees from7-33-97 and PR107 were studied,by quantitative PCR,the 16 genes were screened for spatiotemporal expression,screening for genes specifically expressed in male flowers,analysis of in situ expression using mRNA-FISH,locating and analyzing the selected genes in different tissues of male flowers,to determine where it is expressed in male flowers,cloning of key genes related to male sterility,construction of expression vectors and bioinformatics analysis,at the same time,4 key genes related to male infertility and 6genes of APX gene family were physically located.This study can provide research materials and scientific basis for exploring the mechanism of male sterility in rubber trees,The main results are as follows:1.By analyzing the gene expression patterns of 16 genes related to flower development in different tissues and organs of rubber trees and male flowers at different developmental stages,seven highly expressed genes in male flowers were screened,CCR,UND,AHL16,GEX1,PME53,PME53 and ZAT5.Included the expression of CCR,AHL16,GEX1,PME53 and ZAT5 were up-regulated in the sterile varieties of rubber trees,the gene UND and MYB26 were down-regulated in the sterile varieties of rubber trees,of the four selected varieties.The expression level of these genes in male flowers of rubber tree was higher than that of skin,leaves,female flowers and latex.Their expression is also different between fertile and sterile varieties,indicating that these genes have this tissue specificity in sterile varieties of rubber trees,it has an important effect on the male sterility of rubber trees.2.The analysis of the expression trends in three different developmental periods(microspore mother cell period,tetrad microspore period,mononuclear flower period)showed that the expression of CCR,UND in these three stages showed a tendency to increase first and then decrease,and had a high expression level in tetrad period,indicating that they played an important role in the development of male flowers in tetrad period.At these three stages,the AHL16 expression of these genes in fertile male microspore mother cells were higher,and the expression of these genes were higher in sterile male microspore mononuclear stage,indicating that these genes played a role in the development of male flowers in sterile line and in the development of male flowers in sterile line.At these three stages,the GEX1 showed a decreasing trend of expression in fertile group and sterile group.The expression level of genes in fertile male microspore mononuclear period was higher,and the expression level in sterile male microspore mother cell period was higher.This indicates that these genes play a role in the development of male flowers in fertile and sterile microspore mother cells.3.The in situ expression of these four genes was analyzed by CCR、UND、AHL16 and GEX1 using RNA-FISH techniques.The results showed that the gene CCR、UND、AHL16,whether male sterile or fertile,detected fluorescence signals in cell types such as anther wall and microspore mother cells,It is speculated that gene CCR、UND、AHL16 may be mainly expressed in specific cell types such as anther wall and tetrad spores to regulate the development of male flowers.Abnormal expression in these cell types may lead to male sterility in rubber trees.gene GEX1 fluorescence signals were detected in cell types such as tapetum,anther outer wall and microspore mother cells of anther in male sterile and fertile varieties,but in tapetum cells,the fluorescence intensity of male sterile varieties was significantly higher than that of fertile varieties,it is speculated that GEX1 genes may be mainly expressed in specific cell types such as tapetum,anther wall and microspore mother cells to regulate the development of male flowers,and abnormal expression in these cell types(especially tapetum)may lead to male sterility in rubber trees.4.We located CCR,UND,AHL16,GEX1 and six members of the Hb APXs gene family(Hb APX1,Hb APX3,Hb APX4,Hb APX5,Hb APX6,Hb APX7)on the chromosomes of rubber tree fluorescence in situ hybridization(FISH).CCR long arm located on chromosome 6,The percentage distance between the site of the signal and the centromere of the chromosome is 33.43;UND short arm located on chromosome 16,The percentage distance between the signal site and centromere is 16.32;AHL16 located on chromosome18,The percentage distance between the site of the signal and the centromere of the chromosome is 17.33;GEX1 long arm located on chromosome 8,Hb APX1 short arm located on chromosome 4,the percentage distance between the site of the signal and the centromere of the chromosome is 12.51;Hb APX3、Hb APX4、Hb APX5、Hb APX6 and Hb APX7 were located on chromosomes 7,1,2,9 and 3,Are long arms,the percentage distance between the locus of its gene signal and the centromere of the corresponding chromosome is 80.27,24.72,87.19,13.69 and 57.63.The percentage distance between the site of the signal and the centromere of the chromosome is 22.92.All 10 genes are on different chromosomes,It is speculated that these 10 genes may be genetically independent of each other.Physical mapping to determine the specific location and relationship of these genes on chromosomes,It can provide cytogenetic basis for rubber tree breeding and genomics research.5.The CCR,UND,AHL16 and GEX1 genes were cloned in rubber trees and their function and expression were analyzed for CCR,UND,AHL16 and GEX1 in this study The function of four genes in male sterility of rubber tree laid a theoretical foundation,and also provided a theoretical basis for the breeding of rubber tree.To CCR,UND,AHL16 and GEX1 The four genes further analyzed the specific function of the male sterility mechanism,constructed the expression vector of these genes,and provided the material for the further excavation of the function of the key genes.