Construction,characterization And Phenotypic Analysis Of An AmiR-RUS6 Rus4 Double Mutant In Arabidopsis Thaliana

Arabidopsis thaliana RUS4(ROOT UV-B SENSTIVE 4)and RUS6(ROOT UV-B SENSTIVE 6)are both members of the DUF647(Domain of unknown function 647)gene family.In our laboratory,the RUS4 and RUS6 genes were specifically silenced by artificial micro RNA technology to obtain a RUS4 knockdown strain(ami R-RUS4)and a RUS6 knockdown strain(ami R-RUS6),respectively.It was found that ami R-RUS4 plants had indehiscent anthers and severely reduced male fertility;ami R-RUS6 plants also had significantly reduced fertility,but the T-DNA insert rus4 of RUS4 had normal fertility,and subsequent studies showed that the expression of the RUS6 gene was significantly increased in the rus4 mutant compared to the wild type.Thus,there may be redundancy between RUS4 and RUS6 in the regulation of male fertility in Arabidopsis.This thesis focuses on the construction,identification and expression analysis of the Arabidopsis ami R-RUS6 rus4 double mutant.First,the progeny of the RUS4 knockout mutant crossed with ami R-RUS6 were screened and characterized for pure strains,and a pure double mutant ami R-RUS6 rus4 was obtained.The roots、plant height、lateral stems、anthers and angiosperms of ami R-RUS6 rus4 double mutant strains were observed from different angles.The results showed that the root length of ami R-RUS6 rus4 double mutant was significantly reduced compared to the wild type,being only 1/4 of the wild type.Combined with the gene expression analysis related to the root growth hormone pathway of ami R-RUS6 rus4 double mutant,it can be hypothesized that simultaneous knockdown of RUS4 gene and knockdown of RUS6 gene would cause YUC8 and YUC9 gene expression in the YUC family Up-regulation leads to increased root growth hormone biosynthesis,which in turn induces up-regulation of PIN1,PIN2 and PIN7 gene expression,which in turn leads to increased levels of root growth hormone response and distribution.The plant height of the ami R-RUS6 rus4 double mutant was then measured and found to be significantly lower compared to the wild type but with a significant increase in the number of lateral stems compared to the wild type.This suggests that knocking out the RUS4 gene and knocking down the RUS6 gene at the same time leads to reduced apical dominance in Arabidopsis,thereby altering the distribution of auxin and increasing the number of lateral stems in the ami R-RUS6 rus4 double mutant.Subsequent observations of ami R-RUS6 rus4 double mutant anthers revealed a large number of unreleased pollen grains in the anther chamber,a defective secondary thickening of the anther chamber wall compared to the wild type,and a relative reduction in anther shape.Meanwhile the ami R-RUS6 rus4 double mutant had only 6.6% of the pollen germination rate of the wild type and 70% of the pollen tube length of the wild type.Taken together,it is shown that knocking down the RUS6 gene alone leads to reduced male fertility in Arabidopsis,while knocking out the RUS4 gene and knocking down the RUS6 gene exacerbates this phenomenon,and that the RUS4 and RUS6 genes play redundant roles in the regulation of male fertility in Arabidopsis,the RUS4 gene plays a dominant role and these two genes synergistically regulate anther development and pollen maturation in Arabidopsis.