| Rice is one of the most important food crops in the world, more than half of the global population, including almost the entire population of east and southeast Asia, are feed on rice. In China, rice planting area accounting for1/4of food crops’s, and yield for more than half of food crops. The production of rice directly affect Chinese food supply and people’s living standard. China’s population continues to increase, with the development of city construction, land desertification, arable land decrease year by year and the cultivated land demand of other crops, to ensure the supply of rice must increase rice yield per unit. And sterility directly affects the production of rice, but in the process of rice breeding and rice production, always inevitably experience decreased fertility. Combination between different varieties as internal factors as well as high temperature, low temperature, diseases, and so on of many external factors will be more or less affect the sterility of rice, which makes the fertility changes of rice is extremely complex and appears to be no rules to follow.Mutants is the ideal source material for studying gene expression and function, it is one of the current hot topics in the study of functional genomics. In this study, first we get sterility mutant materials, then analysis the fertility reduced reason at cytology level, finally we get the genes function to explain the molecular mechanism. As a result, we isolated to a new rice sterility gene from Q6537mutants and study the pollen sterility gene (PSS1)which have been cloned. The results enrich our understanding of rice sterility. Main research results of this paper were as follows:(A) rice sterility mutants screeningFrom about10,000T-DNA insertion mutants, that we picked up the20stable genetic lines with low fertility that separated by3:1ratio (controlled by a single gene) and7lines in addition to alone with other phenotype variation. On one of these mutants, M3has carried on the map based cloning, results show that it was allelic of rice Extra glume1.(B) genetic analysis of rice trisome sterility mutant line Q6537,from this line we cloned OsMSH4and analysis the gene’s function1. Our lab have been got a rice male sterile mutants Q6537, the mutant was collected from an anther culture mutants population originated from autotetraploid indica/japonica hybrid H3774(H2088×H891). we surveyed419progenies’individual from Q6537and found the phenotypic ratio of Q6537to Q6537M is111to308(roughly1:3)2. We then attempted to obtain the wild type of mutant Q6537M from the progeny of Q6537. As a result,19normal seed setting plants were selected out of1360individuals consisting of34Q6537plant lines. Among their progeny,14of them displayed further fertility segregation fitting to a ratio of3:13. We then surveyed the meiotic cell to inspect chromosome constitution of Q6537. At metaphase I, other than a full set of12pairs chromosome, one extra chromosome presenting either outside the equatorial plate, or, in most situation (over90%of total56spreads), synapsed with a bivalent to form trivalent, we deduced that Q6537is a typical trisomic line, trisomic Q6537make the inheritance accessible of a full sterility mutant Q6537M.4. To map the gene, we crossed the trisomic Q6537as female parent with9311, an Indica variety, to construct a genetically segregating population. Using the molecular markers, further mapped into a60-kb interval, the mapped region contains9recognizable open reading frames (ORFs). One of the ORFs (ORF8) encodes a protein homologous to AtMSH4, in which there is a single nucleotide substitution at codon482(G/C) in the third exon, resulting in an amino acid change from Ala-118to Pro.5. Through the Q6537M meiotic observation, the most obvious defects became apparent at diakinesis when all meiocytes (>200cells) had<12bivalents, mostly at the range of2-4. The remaining are unsynapsed univalents. Subsequently all bivalents and some univalents aggregated on the metaphase I equatorial plane, make the pollens of Q6537M fully empty that stained by iodium potassium iodide.6. OsMSH4is the homologous of AtMSH4gene in arabidopsis, the gene involved in the exchange of homologous chromosomes, the mutations of Atmsh4gene in the arabidopsis make a large number of univalent during meiosis period, it also make the fertility reduced significant but still have some fertility.7. Gene’s quantitative PCR indicate OsMSH4was meiosis specific gene, at the same time, there are no obvious different expression between Q6537M and WT with other meiotic gene at the upstream and downstream of OsMSH4.(C) Analysis the function of PSS1gene that from a semi-sterility mutant line W207-2and found other proteins that interact with PSS1Our laboratory previously got a stable heredity rice semi-sterility line W207-2, and cloned pollen sterility gene PSS1from the line, PSS1is a kinesin, there is a single amino acid change of the protein which make it lose its function, causing part of the meiotic cell appear a small amount of the univalents, makes the pollen exhibit infertile phenotypes, lead to reduced mechanical pressure generated in the pollen sacs, affect the normal anther dehiscence, allowing the semi-sterility phenotype in W207-2, detailed work has been published about this part. Here are the follow-up experiments:1. PSS1-GFP subcellular localization in rice and arabidopsis protoplast cells showed that it mainly locate near and in the nucleus space, where chromosomes and spindles appeared at meiosis stage2. Microtubules combined experiments in vitro show that a single amino acid mutation makes the PSS1protein lost the ability to combine with microtubules.3.In the pssl mutants, no spindle appear observed flaws. The univalents are the main defects, the formation of the univalents may be the result of abnormal chromosome pairing or is caused by abnormal chromosome movement.4. By the RNA interference of the PSS1gene expression, it make the expression capacity of PSS1in the transgenic plant is significantly lower, the phenotypes of the transgenic plants are the similar with the phenotypes of W207-2.5. By using yeasts two-hybrid method, screening with rice panicle cDNA library, we found two proteins are glutamate synthase Glutamine synthetase (GS) and Targeting protein for Xklp2(TPX2) could interact with PSS1. |
