The Flowering Gene Of Oryza Sativa Clone And Function Research

Rice (Oryza sativa) is a very important crop. There have a lot of genes among the transition from vegetative period to reproduction period .We want to find some genes are crucial among this period. If we can get more crucial genes among this period,the transition of this special been controlled with that gene ,less time it is earlier the plant will grow.In this research we aim at the gene among this period at that time SHISHOUBAIMAO is used as research material.There have some research direction which include subtractive cDNA (including down regulating and up regulating) library constructed,sequencing the different clones ,select three gene (OsEMF2-1,OsEMF2-2,OsFCA) to research using rice information in the web,using RNAi and overexpression research the three gene function in Oryza sativa. we hope using this method get more information of rice flowering .The abtained main results are as follows: 1: A suppression subtractive cDNA library of rice(Oryza,sativa L.SHISHOUBAIBAO) shoot apical meristem (SAM) was constructed using suppression substractive hybridization (SSH) method.The 3,4and 5 leaf period and 5,6,and 7 leaf period SAM were used as "tester"and "driver"respectively. Seventeen of 1920 cDNA clone were identified to down-regulate flowering.Sixteen of 1920 cDNA clone up-regulated flowering. Dot hybridization identified there had 100 true clones in upregulated cDNA library at the same time there had 48 true clones among the downregulated cDNA library.There had 148 clone needed to next identify. 2:After reverse Northern there had 28 different clone in the upregulated cDNA library and 25 clones in the downregulated cDNA library.Using HaeII digest the different clones and classfied the same clone using the same digest band. 3:The cDNAs were cloned into pMD18-T vecor.17 clone have been identified by screening with forward and reverse subtracted cDNA probe. When 3+4+5 leaf period was "tester".and 16 clone were identified when 5+6+7 leaf period was "tester".that is ,we got 33 clone in different period including 16 clones of up-reglated and 17 clones of down-regulated. All the thirty-three cDNA were sequenced (Bioasia CO LTD).The information of the prediction protein were got from the RGP and the NCBI. 4:Test whether the selected 33 clone are truly expressed differential in downregulate and upregulate to flower. We randomly choose 10 clones respectively and used the inserts as probe for Northern hybridization with total RNA in different period,the signal of expression is decreased from different leaf period .that is this clone is a true differentially expressed gene.. Only one of the 10 down-regulating clone had hybridization signals while four of the 10 up-regulating had true signals.There had 25 percent clones which have Northern signals. The rest clones which had no Northern signals were used realtime PCR.Most of the clones are true expression gene. 5:Our important research is in these three gene OsEMF2-1(embryonic flower2-1 ), OsEMF2-2 (embryonic flower2-2 )and OsFCA(flower time control gene),The function of the three gene in the rice flowering revealed by RNA interfence vector(our lab no published). Especially the OsEMF2-1,except RNA interference we also constructed overexpression vector(our lab no published). 6:RT-PCR result indicated the three gene all expressed in the panical,they all no expressed in the root,because these gene all connected with flowering they expressed in the panical is essential. 7: Southern hybridization of the thansplant T0 HPT as probe most of the the transgenetic plant T-DNA copy were 1-3 different copy. The transfer were succeed. 8:There had a 1kb transcript produced after RNA interference while in the negative CK and other transgene plant no that transcript. After RNAi there had a new transcript produced. 9: The OsEMF2-1 overexpression plant no signal after Northern blotting except one transgenetic plant had two different 5kb transcript using OsEMF2-1 cDNA as probe.Which Northern signal was Nos transcription. 11:The pollen of the transgenetic plant is normal as the negative plant.The overexpression T0 transgenetic plant of OsEMF2-1 had strange phenotype of pollen,the most pollen were gametic sterility.Maybe the function of OsEMF2-1 was connected with the development of pollen...