Cloning And Functional Analysis Of Seed Vigor Related Genes In Rice(Oryza Sativa L.)

Rice(Oryza sativa L.)is one of the most popular crops in the world.Direct seeding of rice at present is popular in many countries because of its low cost and convenience compared to the conventional transplantation.In direct rice production,seed germination and seedling formation time may be delayed due to adverse environmental factors of rice surface or puddle soil,and seedling mortality is greatly increased and yield loss risk is increased.Seeds with high vigor may significantly improve the speed and uniformity of seed germination and the final percentage of germination,and lead to perfect field emergence,lead to better suppression of weed growth,and produce high yield under different conditions.Therefore,the mining of key genes regulated seed vigor and illuminating their mechanisms are important objectives of rice breeding for direct seeding production.In this study,the rice seed vigor related genes qSV3 were cloned.Main research findings are as follows:(1)We used 62 CSSLs developed by introgressing chromosome segments of Jiucaiqing into IR26 for QTL mapping.The seed germination percentage(GP),germination index(GI)and seedling percentage(SP)of 62 CSSLs were assessed under H2O condition respectively.By single marker analysis(SMA),we identified seven QTLs,qGP3.1,qGI3.1,qGP3.2,qGI3.2,qGP10.1,qSP3.1 and qSP10.1 for GP,SP and GI,respectively,and their positive alleles were derived from IR26 with an increase of seed vigor,except qGP3.1 and qGI3.1.The qGP3.2,qGI3.2,and qSP3.1 were mapped to be tightly associated with marker RM3513 on chromosome 3,integrated as a qSV3 locus.The qGP10.1 and qSP10.1 were mapped to be tightly associated with the marker RM5348 on chromosome 10,integrated as a qSV10 locus.Since the qSV3 showed higher contribution to seed vigor with 37.75%of average phenotypic variation(APV)of GP,21.74%of APV of SP,and 88.52%of APV of GI,it was further analyzed in this study.(2)The residual heterozygous line CSSL10 containing the allele(qsv3)from parent Jiucaiqing with the low GP,SP and GI,was selected for confirming qSV3 locus.A segregation population consisting of 120 individuals was developed by self-fertilization of CSSL10,and their GP at 2 days after imbibition showed bi-modal characteristic distribution,suggesting the presence of a major gene.Inclusive composite interval mapping(ICIM)showed that there was a significant QTL of GP between markers HY27 and HY44,with 55.48%of phenotypic variation.5,000 individuals from the BC6F4 population were used to narrow down the qSV3 locus into a genomic region between the markers RM135 and HY44.992 individuals of the BC6F5 population were used to further delimit the qSV3 locus.(3)The qSV3 locus in a 6.5 kb region that contained two predicted genes.Sequence variation analysis indicated that seven single-base substitutions occurred in the coding region of OsHIPL1 between Jiucaiqing and IR26,of which five substitutions led to amino acid substitutions.Expression analysis showed that the expression of OsHIPL1 was significantly higher in IR26 than in NIL-qsv3 in germinating seeds under H2O condition.By used CRISPR/Cas9 system to construct Oshipl1 mutants,performed a genetic complementation test in the NIL-qsv3 background and generated OsHIPL1-overexpression lines,OsHIPL1 was finally considered as a causal gene for qSV3.OsHIPL1,which encodes hedgehoginteracting protein-like 1 protein,a new gene of rice seed vigor.(4)By the quantitative RT-PCR(qRT-PCR)approach the expression patterns of the OsHIPL1 gene were analyzed in various tissues,and germinating and developing seeds in IR26.There was a relatively higher expression of OsHIPL1 identified in the root,compared with that in the stem,leaf,sheath,internode and spike.During seed germination the transcript levels of OsHIPL1 were gradually increased at the early stage(0 h～30 h after imbibition),then decreased at the late stage(36 h～72 h after imbibition).During seed development the expression levels of OsHIPL1 were gradually increased from 0 to 35 days after flowering.To determine the subcellular localization of OsHIPL1,a recombinant OsHIPL1 protein tagged green fluorescent protein(GFP)at the C terminus under the control of the 35S promoter was constructed and expressed transiently in rice protoplasts and N.benthamiana leaves.The GFP-tagged OsHIPL1 was found to be mainly localized in the plasma membrane and nucleus in rice protoplasts and N.benthamiana leaf epidermal cells.(5)To explore the regulatory mechanism of OsHIPL1 on seed vigor,the RNA-seq analysis was performed between IR26 and NIL-qsv3 at the 12 hours after imbibition.A total of 3,186 differently expressed genes(DEGs)were identified,including 1,432 up-regulation and 1,754 down-regulation,in NIL-qsv3 compared to IR26.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway showed that differently expressed genes involved in phenylpropanoid biosynthesis,plant hormone signal transduction,Brassinosteroid biosynthesis and Starch and sucrose metabolismetc.The many DEGs between IR26 and NILqsv3 were involved in the plant hormone signal transduction includingabscisci acid(ABA),auxin(IAA),gibberelin(GA),brassinosteroid(BR),ethylene(ET),cytokinin(CK)and jasmonic acid(JA).The higher levels of endogenous ABA were measured in germinating seeds of Oshipl1 mutants and NIL-qsv3 line compared to IR26 plants,with two up-regulated ABA biosynthesis genes(OsZEP and OsNCED4)and one down-regulated ABA catabolism gene OsABA8ox3.The expression of ABSCISIC ACID-INSENSITIVE 3(OsABI3),OsABI4 and OsABI5 were significantly up-regulated in germinating seeds of Oshipl1 mutants and NIL-qsv3 line compared to IR26 plants.These results indicate that the regulation of seed vigor of OsHIPL1 may be through modulating endogenous ABA levels and altering OsABIs expression during seed germination in rice.(6)To further understand the molecular mechanism of regulation by OsHIPL1 on seed vigor in rice,we identified the interacting proteins through a yeast two-hybrid assay with OsHIPL1 as bait.Of the identified 22 candidates,4 candidate proteins were subsequently confirmed by retransformation into yeast,including aquaporin OsPIP1;1.To further confirm the OsHIPL1-OsPIP1;1 interaction,the bimolecular fluorescence complementation(BiFC)and luciferase(LUC)assays were conducted.The yellow fluorescent protein(YFP)signals were only observed on the plasma membrane of N.benthamiana leaves when cYFPOsHIPL1 was co-infiltrated with nYFP-OsPIP1;1.Meanwhile,only co-expression of cLUCOsHIPL1 and nLUC-OsPIP1;1 in tobacco leaves could reconstitute LUC activity compared with the various negative controls.These results demonstrate that OsHIPL1 could interact with OsPIP1;1.To investigate whether OsHIPL1 affects water uptake during seed germination,we tested the water content of the germinating seeds.The water content of Oshipl1 mutants was significantly reduced compared with that of IR26.By contrast,the water content of COM and OE lines was significantly increased compared with that of NIL-qsv3 line and ZH11,respectively.The OsPIP1;1 expression was also tested.A reduction of the transcript levels of OsPIP1;1 in Oshipl1 mutants was observed compared with that in IR26,and an increase of the relative expression levels of OsPIP1;1 in COM lines was observed compared with that in NIL-qsv3 line.Thus,we speculated that OsHIPL1 interacted with the aquaporin OsPIP1;1,then affect water uptake to promote rice seed germination.(7)To identify the allelic constitution of OsHIPL1,192 rice accessions of the Rice Diversity Panel 1(RDP1)were selected to be evaluated by GP,SP and GI during seed germination.We identified a Hap1 haplotype of OsHIPL1 that was positively correlated with seed germination.To investigate the application of OsHIPL1 for direct seeding,seedling establishment and seedling growth were compared among IR26,Oshipl1 mutants,NIL-qsv3 and the COM lines,and ZH11 and OE lines when seeds were directly sown in soil.In the greenhouse,the SP,root length,shoot length and basal shoot diameter were significantly higher in IR26 than in NIL-qsv3 line and Oshipl1 mutants.Moreover,the COM lines exhibited a significant increase in the SP,shoot length,shoot length and basal shoot diameter compared with those of NIL-qsv3 line,and the OE lines significantly increases in the shoot length and basal shoot diameter compared with those of ZH11.Meanwhile,there were the decreases of the SP of NIL-qsv3 line and Oshipl1 mutants compared with those of IR26 and the increases of the SP of COM lines compared with those of NIL-qsv3 line under the field.It is beneficial to facilitate improvement of seed vigor in rice breeding program and beneficial to production of rice direct seeding.Summary,we clarified the function of hedgehog-interacting protein-like 1 protein gene OsHIPL1 in plants.There are two possible pathways by which OsHIPL1 regulates seed vigor in rice.On the one hand,OsHIPL1 contributed to seed germination and seedling establishment involved in modulating ABA-1 evels and signaling in germinating seeds in rice.On the other hand,OsHIPL1 interacted with the OsPIP1;1,and regulated the water uptake to improve rice seed vigor.Our studies provide a novel gene OsHIPL1 and its information on the molecular mechanisms of seed vigor.Meanwhile,the superior haplotype HAP1 and its linkage molecular marker SNP3 of OsHIPL1 were identified,which would facilitate improvement of seed vigor in rice breeding program and beneficial to production of rice direct seeding.