The Study Of Oscbl5 Gene Regulation GAs Signaling Pathway In Rice Effects Of Pollen Fertility

In eukaryotes,Ca+ is the most important Second messenger in signal transduction,calcineurin B-like proteins(CBLs)is a kind of special calcium ion sensor which has been identified in recent years,At present,the research on the function of CBL5 gene is mainly focused on stress:salt stress,drought stress and so on.Through the resent years study,we have found that cbl5 mutants type plant height and seed setting rate was significantly lower than the wild type Hwayoung.So,we inferred that cbl5 gene may also controlling plant height and pollen fertility.The laboratory have people research in-depth about OsCBL5 gene regulation of pollen fertility,and do the preliminary discussion about it effection on plant height.This experiment mainly make in-depth research on OsCBL5 gene effects about plant height of rice.As is know to all that Plant height is one of the important agronomic traits of rice,which has a direct relection on the yield of rice.If the plant is too high,proned to lodging,leading to cut,and the excessive dwarfing will lead to reduce the rate of energy utilization.In the fifties of the last century successfully bred of semi dwarf varieties,these species not only resistant to lodging,also makes the world rice production reached an unprecedented breakthrough,so appropriate reduce rice height is also very necessary.The materials of this study were the mutant OsCBL5 and the wild type Hwayoung,Mutant OsCBL5 was mutated by T-DNA insertion.The key genes of the GA signal pathway synthetic pathway were quantitative analyzed.And the content of endogenous GAs was detected,and the compensation of gene function was constructed to restore the vector transformation.,constructed transient expression vector,the main result were followings:(1)Agronomic characters analysisThe resent two years of agronomic characters of mutant rice and Hwayoung have been analysis,we found that the average height of mutant oscbl5 was only 34 cm,lower than Hwayoung 73cm,the mutant plant germination rate is only 12.5%,but Hwayoung higher than 87.7%,contrast the tassel length of mutant plant oscbl5 and Hwayoung,we found that the tassel length of mutant plant oscbl5 is only 10.6cm,shorter than Hwayoung 17.2cm.The results of Agronomic Traits in two years were consistent with previous.It is proved that the agronomic characters of mutant oscbl5 can be stably inherited.(2)The pollen fertility of mutant oscbl5 and wild type rice Hwayoung was statistically analyzed by K-KI staining method.The results showed that the pollen contamination rate of mutant oscbl5 was significantly lower than that of the wild type Hwayoung,the deep dyeing rate of Hwayoung was 85.2%,and the rate of oscbl5 was '12.8%.The results are consistent with previous experimental results.(3)Analysis of seed germination rateThe germination rate of the oscbl5 and Hwayoung rice seeds of the first day to the fourth day was observed.The germination rate of oscbl5 mutant was lower than that of wild type Hwayoung.On the second day of the seed germination,the germination rate of the oscbl5 mutant was only about 10%,while the Hwayoung reached 30%.During the fourth days of seed germination,the germination rate of Hwayoung was 72%,and the mutant was only 50%.(4)Construction of restoration vectors and transformationIn this study,the OsCBL5 gene expression vector was constructed by enzyme digestion method and transformed into the rice,Through PCR,Gus staining,fluorescence quantitative PCR method to verify the positive strain of transgenic plants,found that the plant height of transgenic plants was restored to the wild type,and the seed setting rate was increased.(5)Subcellular localization analysisIn this study,we use the gateway mathod to construct OsCBL5's transient expression vector.Analysis of subcellular localization,Under the fluorescence microscope,it was found that the gene was mainly localized on the plasma membrane.The experimental results verify the previous results.It showed that the gene may have a certain effect on the signal transduction pathway of GA.(6)Determination of the content of endogenous GA in Rice The content of endogenous GAs in mutant plants and wild type Hwayoung was detected in this experiment.14 kinds of GAs were selected,and the detection results were analyzed.We found that the total content of the 14 kind of GAs in the mutant was higher than that in the wild type,but the content of GA with biological activity was lower than that of wild type.(7)Analysis of gene expression of key enzymes in GA biosynthesis pathwayTo detected the expression of key enzymes in the pathway of GA biosynthesis of mutant plant and Hwayoung have found that the relative expression of CPS,KS,KO and KAO of mutant plant oscbl5 is lower than Hwayoung,the relative expression of CPS only about 32.4%of Hwayoung,and KS only about 57.4%,KAO only about 69%,GA20ox only about 38%.The result showed that the expression of key enzyme gene in the pathway of GA obvious different compare to Hwayouung.(8)Quantitative analysis of key genes in the signal transduction pathway of GAThe study have tested relative expression of GID1,GID2 and RGA witch is important for GA signal transduction pathway,found that the expression of GID1 and GID2 in mutant rice was lower than that in wild type.The relative expression of GID1 was only 11.4%of the wild type.The relative expression of GID2 was only 22.1%of the wild type.Analysis of theexpression of negative regulation factor RGA found that the mutant was higher than the wild type,was about 130.1%of the wild type.these results indicate that the GA signal transduction pathway of mutant plants may also be blocked.(9)Analysis of the expression of CIPKs geneIn order to further study the mechanism of action of OsCBL5 gene,The expression levels of CIPKs gene were analyzed and the results were found that,In OsCBL5 mutant plants,CIPK1 and CIPK3 up regulation,and CIPK11,CIPK17,CIPK18,CIPK23,CIPK26 expression is down,this part of the results can be used for the latter part of the study.