Full-Length CDNA Library Construction And Functional Analysis Of Two Micrornas In Rice

Rice(Oryza Sativa L.) is one of the most important crops worldwide.Identification of genes,which control plant height,heading date,number of tillers and panicle size,is essential for rice genetic improvement.Endogenous small RNAs,especially microRNAs, are key regulators for plant development.MicroRNAs are novel candidate genes for rice genetic improvement.There are three objectives of this research:1) construction of cDNA libraries including full-length cDNA libraries and yeast two hybrid cDNA libraries;2) identification and analysis of the OsSPL genes from rice,specifically for miR156 targeted OsSPL genes;and 3) studying the functions of miR156 and miR164 in rice.In the first part,the main results are as follows:1.One full-length cDNA library has been constructed from young panicles(stage 3, 4,5).The sequences of 3,000 random selected clones indicated that more than 92% clones are full-length cDNA.A total of 10,828 cDNA clones,which contain full length open reading frames,have identified from the conventional cDNA library.Thus,13,650 full-length cDNA clones have been identified in this research.2.Two cDNA libraries have been constructed for yeast two hybrid(Y2H).The cDNA library have been used for Y2H screening for identification of interacting proteins of intested proteins in our group with less than 10%false positive clones.The main results of the second part of this research:1.OsSPL gene family in rice was systematically analyzed.18 OsSPL genes have been identified.The phylogenetic relation ship of plant SPL genes,conserved protein motifs,and expression pattern in 13 different tissues were studied in this research.2.There are 11 OsSPL genes containing M156BR(miR156 binding region) based on sequence analysis.The miR156-directed mRNA cleavage of OsSPL12 and OsSPL14 can be detected when they were over expressed in plants.3.The cleavage sites of OsSPL14 and OsSPL17 were mapped by RLM-RACE. The cleavage sites of miR156 targets were located between the seventh and eighth nucleotide of M156BR,where is the mismatch site of M156BR and miR156.4.Two point mutations of OsSPL14 gene,OsSPL14m1 and OsSPL14m2,were generated and OsSPL14m2 was over-expressed in rice.5.OsSPL14-interact-proteins were identified by Y2H screen of a panicle cDNA library.Three of them encode a RING finger protein,which is involved in ubiquitin pathway,suggesting that OsSPL14 may involved in the regulation of protein turnover.In the third part of this research,the functions of miR156 and miR164 was analyzed. The results list as follows:1.Two precursors of miR156(pri-miR156d and pri-miR156h) and one of miR164 (pri-miR164b) were transformed rice under the control of maize ubiquitin promoter.The transgenic plants named Md(pri-miR156d),Mh(pri-miR156h),and MI7(pri-miR164b) respectively.2.The developmental difference of Md and Mh were investigated.Md and Mh plants showed distinct developmental differences to WT after the fourth leaves emerged. The rate of leaf and tiller initiation of Md and Mh are 300%of that in WT.The heading dates of Md and Mh delayed more than 7 days.Compared to WT,the number of leaves increased more than one hundred folds and the number of tillers increased more than 50 folds.However,the plant height of the Md and Mh plants decreased 50%.Though the branches of panicle and the spikelet number of Md and Mh decreased dramatically,the yield and biomass are not changed.The shape and epidermis of leaves indicate that the developmental time of rice was changed in Md and Mh.3.The expression level of miR156 increased gradually parallel to the development of leaves.It is concluded that the expression level of miR156 can reflect the developmental time of rice leaf as a marker gene.4.The up-and down-regulated genes in mature or immature leaves of Md and Mh were identified by microarray assay.5.The interaction of miR156 and OsSPL may be regulated by other regulators.Two candidates including a no-coding RNA gene(DRG12) and PLA2 were identified in this research. 6.The expression of miR164 showed reverse pattern to miR156 during the leaf development.An unknown primary transcript of miR164 has been identified in small RNA gel blot.It is suggested that the expression of miR164 is regulated by its promoter and microRNA processing.7.miR164 targets OsNAC1 and OsNAC2 in rice.Cleavage site mapping indicated that miR164 cleavages the 10^th nucleotide of M164BR.The transcript levels of OsNAC1 and OsNAC2 were decreased when miR164 was increased in different leaves.8.miR164 may regulate the organ boundary.The defects of MI7 leaf include fused leaf sheath and twist leaf blade.At reproductive stages,the abnormal development of MI7 pistil turns out sterile.9.The defects of MI7 can be rescued by applying hormones.The genes involved in auxin synthesis,transport and responses were down-regulated in MI7.Moreover,DRG12 is down-regulated in MI7.10.miR164 plays an essential regulation role in rice reproductive growth.One of the internodes of MI7 main culm did not elongate.In MI7 leaves,a LFY/FT homologous gene(MI7D1) was down-regulated.In addition,the expression levels of OsNAC1,OsNAC2,and MI7D1 were changed in association with photoperiodic rhythm: all the genes showed peak expression in the middle of dark phase.11.The expression pattern of miR156-miR164 is conserved in gramineae crops, but different in Brassica napus.12.A paradigm model of miR156-miR164 action has been concluded to explain the regulatory role of miR156 and miR164 in rice.