Preliminary Functional Analysis Of A Male-female Fertility Controling Gene In Rice

Rice is one of the world’s major food crops, accounts for about50%of the world’s population for the staple food. Sterility of rice not only affects the level of production, and directly related to the approach of heterosis’ utilization, so the rice fertility research has theoretical significance and practical value. With the completion of the sequencing of the rice genome,EST libraries,the mutant library and the development of gene expression profiling technology, a lot of rice fertility-related genes have been cloned and described functions, progress has achieve about rice infertility molecular mechanism.In this study, mutant material MFS-z9is found in the transgenic T2progeny of ZH9, the mutant is sterile both male and female. The early studies have shown that the mutant male sterile because of tapetum do not degradation during meiosis, it lead to pollen sterility; female sterility due to the embryo sac undifferentiation or differentiation is not complete. Gene fine mapping showed approximately20K mutant DNA have deleted (containing three gene ORF1, ORF2and ORF. We finished cloning, analysis of candidate gene and expression vector construction of candidate gene ORF1and ORF3, In this study, we have the job of rice genetic transformation by agrobacterium-mediated genetic systems, and get the positive plants after PCR molecular detection, phenotypic analysis of each positive plants and candidate gene expression patterns were analyzed. The main results as follows:1Genetic transformation of functional carrier Let the candidate genes ORF1and ORF3over-expression, RNAi interference, antisense expression vector into embryogenic callus by agrobacterium-mediated genetic transformation system. We get92resistant seedlings for ORF1over-expression vector,268resistant seedlings for ORF1RNAi interference vector and78resistant seedlings for ORF1antisense expression vector vecto. We also get135resistant seedlings for ORF3over-expression vector,300resistant seedlings for ORF3RNAi interference vector and116resistant seedlings for ORF3antisense expression vector vecto. It is ORF1and ORF3complementary candidate gene expression vector that introduced into Young panicle callus by agrobacterium-mediated genetic transformation system, the resistant calli were obtained from311and579pieces;162and264strains resistant seedlings obtained. 2Positive plants of transgene obtained Molecular detection by PCR, Basta and hygromycin resistant detection, we get26positive plants for the ORFlover-expression vector,10positive plants for the RNAi interference,3positive plants for antisense expression vector and17positive plants for the complementary expression vector, we get30positive plants for the ORF3over-expression vector,10positive plants for the RNAi interference,2positive plants for antisense expression vector and11positive plants for the complementary expression vector.3Phenotypic characterization and functional analysis The positive plants fertility traits were not restored after the ORF1complementary test. RT-qPCR evealed that ORF1positive plants have been fully expression, while positive plants seed setting rate do not change in RNAi interference. We speculated that ORFl control the gene for infertility traits. The positive plants mutant traits were not restored after the ORF3complementary test. QPCR revealed that the expression of ORF3in wild-type was significantly reduced, we analyzed the reduce of expression of ORF3in positive plants leading to fail of complementary test, but after RNAi interference, the continuous strong seed setting rate cut down in TO and T1of positive plants, seed setting rate and expression are up very relationship, so we initially speculated that ORF3control the gene for infertility traits.4Expression pattern of candidate gene We detect different tissue stages of ORFl, ORF2and ORF3flowering period in Nipponbare by RT-qPCR. According to the expression of three genes indifferent tissues, the three candidate genes are constitutively expressed gene. But they distribut in the various tissues with the developmental stages of dynamic changes:The ORF1is constitutively expressed in anthers before meiosis, mainly expressed in the roots and stem in meiosis; The ORF2expressed in spike before meiosis, expression of ORF2drastically reduceed and subsequently expressed in stem and spike in meiosis; The ORF3is constitutively expressed in anthers before meiosis, mainly expressed in the leaf and spike in meiosis, and expression in spike declined sharply in meiosis anaphase, And expression has advantages in leaf. Taking into account the reproductive disorder of mutant occurs mainly in meiotic prophase, consistenting with a high degree of ORF3genes and their functions.