Cloning And Application Of A Senescence-specific Promoter In Rice Leaf & Identification Of Early Senescence-associated Genes In Flag Leaves

Rice is one of the most important food crops in the world.Though the spread abroad of hybrid paddy rice greatly resolved the lacking of food for the world population,most of the wildly-used hybrids(especially indica rice) were early senescence in the late development,which limits the sythesis and accumulation of carbohydrates,unfavorable to seed rating or grain yield improvement.The key to solve the problem is to understanding the mechanism of leaf senescence and then find a pathway to delay senescence,so as to improve grain filling.Utilizing cytokinin synthesis gene under control of senescence-specific promoter,suppressing senescence up-regulated genes or overexpression senescence down-regulated genes all possibly lead to creating stay-green rice.In this study,a rice senescence-specific promoter was isolated and identified,and drived a cytokinin synthesis gene from Agrobacterium in transgenic indica and japonica rice,then the stay-green lines were selected.At the same time,we constructed a flag leaf early senescence SSH library of japonica rice Zhonghua 11.The main results were as follows:1.A rice promoter P_SAG39 was cloned and its time and spatial expression patterns were identified in transgenic plant through the repoter gene gus.Gus staining results showd the promoter expressed in leaf,culm,root,flower,glume;immature seed coat and callus,but not in mature seed.mRNA of the gene accumulated at highest level at late senescence stage.Above all,the promoter is senescence specific and could be used in crop genetic improvement.2.According to PLACE,various cis elements were predicted.We checked expression mode of a series of 5'-truncated promoters in response to hormones and cold treatment in rice calli,and to leaf senescence in 5' end deletion transgenes.In the analysis of gel retardation assay,we obtained two cis-acting elements HBOXCONSENSUSPVCHS and WRKY71OS for responding to leaf senescence.3.P_SAG39:IPT was transformed to japonica rice successfully.We selected homozygous IPT transgenic lines with single copy insertion,containing 5 lines using MDJ8^# as a recipient named MT1,MT2,MT3,MT4 and MT5,and 3 lines using ZH11 as a recipient named ZT1,ZT2 and ZT3.4.Measuring chlorophyll concentration of flag leaves indicated that stay-green phenotype was coseparation with the leves of ipt in P_SAG39:IPT positive transgenes. 5.We investigated the impact of cytokinin in the transgenes on seedling growth and heading mediated by the lighta and found that in a certain light conditions(14 hours or 24 hours) cytokinin has affected the germination of seeds,the growth of buds,but not the stem elongation.In the long-day conditions,cytokinin stimulates the expression of OsMADS50,thereby leads to early flowering.6.The changes of carbon and nitrogen metabolism were detected in ZT1 at reproductive growth stage.By sugar and nitrogen contents determination,we found sink source transition triggered the onset of leaf senescence.7.The plot-test was performed to evaluate green-stay and other agronomic traits by random block designation in triplicates.In ZT1 and ZT2 the biological yield decreased about 8-10%,and 1000-grain weight increased about 5-9%compared with that of the non-transgenic plants.8.We constructed a flag leaf early senescence library of japonica rice Zhonghua 11 by suppression subtractive hybridization using the green flag leaves as the driver and the early natural senescence flag leaves as the tester.815 differentially expressed ESTs were screen using total RNA of driver and tester as probes through macroarray analyses.A total of 533 unigenes have been confirmed by bioinformatics analysis(ESTClustering and GO annotation).183 of these unigenes have GO annotations,involved in macromolecule metabolism,protein biosynthesis regulation,energy metabolism,gene expression regulations,detoxification,pathogenicity and stress,cytoskeleton organization and flower development.Another 121 unigenes co-localized with previously reported known stay-green QTLS.Else were functional unkown.9.To confirm the efficiency and accuracy of the library,we performed reverse northern blotting analysis using 50 randomly selected cDNA fragments as probes from the SSH library.The result showd the efficiency was more than 60%.RT-PCR analysis on the other novel genes indicated that they can be up-regulated in natural early senescence and induced by hormone in different levels.These genes provide new insight into the onset of leaf senescence mechanism.