Cloning And Functional Analysis Of Male Sterility 92 Which Controls Anther Development And Yellow Leaf And Dwarf 1 Which Regulates Leaf Senescence In Rice

1.Cloning and Functional analysis of Male Sterility 92 Which Controls Anther Development in RiceBoth cytoplasmic male sterility(CMS)and nuclear male sterility are a result of abnormal sexual development of anther and/or pollen grains.The pollen development depends on not only involved in the formation of pollen wall by itself,but also providing nutrients for pollen and later the degradation of the tapetum cell in the anther,which is considered to be triggered by programmed cell death(PCD).However,both of them participation in the molecular mechanism of pollen development remains poorly understood.A spontaneous male sterile mutant in Indica cultivar rice,SC-ms-2,was obtained from the F4 progeny of the cross between D297B and Changfeng B.Genetic analysis indicated that male sterility of SC-ms-2 was controlled by a single recessive gene.Map-based cloning and biological functions of this MS92 gene has been studied in this study.The main results are as following:1.1 The rice male sterile mutant SC-ms-2 exhibits no obvious defect in vegetative growth,and the male sterility is not affected by light and temperature.Histology section analysis indicated that the mutant exhibited aborted degradation of tapetum and defective of pollen wall.1.2 The MS92 encodes a putative PHD finger protein,which is the rice homologue to Arabidopsis MALE STERILITY(MS1)gene.Our results confirmed replacement of a 1036 bp DNA fragment by 25 bp nucleotides in the promotor of MS92 resulted in undetectable its expression in SC-ms-2 as evidenced by RT-qPCR analysis.Over-expression of the MS92 caused fertility reduction and plant dwarf.1.3 The results from in situ hybridization analysis showed that MS92 was expressed in tapetum and microspores at the microspore stage.The nuclear localizations of MS92-eGFP fusion protein in onion epidermis cells and Nicotiana benthamiana leaf cells were consistent with the notion that MS92 is a putative transcription activator,and a transcriptional activation assay in yeast indicated that the activation domain is PHD finger module of MS92.Transcript sequence was performed on the florets between WT and SC-mss-2 at the microspore stage.Mis-regulations of phytohormones signals,transcription factors and tapetum cell death led to abnormal anther development in SC-ms-2.1.4 OsEMF2b mediated the protein interaction between MS92 and PRC2 complex.These results indicated that MS92 is essential for the tapetum and pollen wall development.Understanding the relationship between tapetum PCD and pollen wall development regulated by the MS92 has important biological significance,as well as establishes gene and technical foundation for the artificial modification and innovation of new sterile resource.2.Cloning and Functional analysis of Yellow Leaf and Dwarf 1 Which regulates Leaf Senescence in Rice Senescence is essential for the lifespan of plant,as well as a consequence of natural selection for plant reproduction.Leaf is the main organ where produces photosynthate.Early senescence of leaf has significant effects on the grain filling and harvest index in the rice and other crops,which restricts the crop yield potential and even causes dropping in quality.The yld1 mutant with decreased plant height and yellowish leaf was previously identified from the mutant library,which was generated from heavier panicle hybrid rice cultivar Shuhui527 using EMS.Genetic analysis indicated that these phenotypes of yld1 mutant were controlled by a single recessive gene from the F2 populations,which were crossed between these mutants with Indica varieties.This study is proposed on these bases and significant progress has been made in gene cloning and biological functional verification.The main results are as following:2.1 The yld1 mutant did not become yellow compared with WT until the seedlings developed into the tillering stage;The tip of flag leaf began to turn yellow as soon as flowering,which was 10 days earlier than WT;Semi-section analysis suggested that the plant height of yld1 was reduced as a result of each shortened internode;Except the declining of the value of chlorophyll a/b,an ultrastructural study of yld1 chloroplasts in flag leaf revealed abnormal grana organization and starch accumulation compared with that of WT.2.2 Mut-MAP based on re-sequencing and bioinformatical analysis results showed that YLD1 was distinguished from all the other genes related premature leaf,which encoded a P4-ATPase.Sequencing this gene in yld1 showed that a single nucleotide G substitution A in the putative tenth exon,causing neutral glycine(G)into acid aspartic acid(D)in the key motif DGET of ATPase which is the highly conserved in all organisms,possiblely resulting in a lack of ATPase activity.2.3 The results from RT-qPCR showed that YLD1 was constitutively expressed in rice,while the senescence degree of flag leaves was positively correlated to the expression of YLD1 after the heading stage.The YLD1-RNAi transgenic lines(T0)displayed leaf yellow and plant dwarf.2.4 The glycolipids such as monogalactosyl diacylglycerol(MGDG)and phospholipids such as phosphatidylcholine(PC),lyso-phosphatidylcholine(LPC)and lyso-phosphatidylethanolamine(LPE)were reduced in the flag leaves at the 5 days after flowering.The expression of senescence up-regulated genes were either reduced or increased,while the expression of several genes encoding components of the light harvesting chlorophyll binding protein(LHCP)complexes was decreased in the flag leaves at the 10 days after flowering in yld1.2.5 Salicylic acid(SA)concentration was significantly increased in the flag leaves at the 5 days after flowering between WT and yld1,while any difference was found among IAA,ABA and JA level compared with WT.Detached leaves of yld1 at the tillering stage were more sensitive to SA and exhibited accelerated senescence compared with WT when incubated with 100umol/L SA under normal light.These results suggested that YLD1 is crucial for leaf senescence in rice,especially nutrient recycling of aged flag leaf after the heading stage.These data will shed light on the importance of P4-type phospholipid flippase in leaf senescence and provide theoretical basis for crop yield increase and quality improvement.