Cloning And Study On The Function Of Protein Kinase C Gene In Rice

Rice is one of the most important food crops in the world.More than half of the world’s people live on rice.With the continuous progress of world industrialization in the 21st century,the reduction of rice planting land caused by environmental problems such as land salinization,lack of fresh water resources,soil erosion and global warming finally seriously threatens the production level of rice.One of the important problems that we need to address in order to further improve the quality of rice under the adverse environment of modern scientific research is how to maintain the stability of rice production.With such a sense of mission,our laboratory found and confirmed that rice catalase C(CatC)can play an important role under salt stress,which provides a new idea for the genetic improvement of rice.As one of the connecting parts of CatC signal network in rice,little is known about the research of rice protein kinase C(PKC),which transmits external signals to the upstream protein of CatC.It has sufficient research value to analyze the role of rice PKC in what kind of stress and what kind of function it plays in stress,which is of great significance for rice to further resist environmental threats.Thus,this study cloned two rice PKC genes Os03g21620 and Os07g48290 from rice c DNA,constructed and obtained the overexpression of these two rice PKC genes and mutant transgenic plants respectively,preliminarily verified the phenotype of their transgenic plants,and further analyzed their possible functional models.The main results are as follows:(1)In order to study the expression pattern of rice PKC,we first cloned two rice PKC genes Os03g21620 and Os07g48290 from the rice genome,and then compared them with human PKC,and found that they have 28.7% and 30.4% similarity respectively.Then we used the website to predict the tissue specificity of the two rice PKC genes,and found that they may have the highest expression in different tissues,The tissue-specific expression was verified by quantitative PCR.Then we analyzed the cis acting elements of the promoters of the two genes using the website,and found that they may be involved in the stress resistance of rice.Finally,we found that their subcellular localization is on the cell membrane of rice protoplast,which may play a role on the cell membrane.(2)In order to further study the function of rice PKC,we constructed two rice PKC gene overexpression and gene editing mutant vectors,and obtained transgenic rice plants through agrobacterium mediated rice transformation technology.After identification,11 overexpression positive plants of Os03g21620 gene and 1 gene editing mutant pure and mutant plants were obtained respectively,While 12 plants with overexpression of Os07g48290 gene and 2 plants with gene editing mutant and pure mutant were obtained.Then,preliminary phenotypic identification was carried out.It was found that rice PKC gene mutant had a high temperature resistant phenotype,and overexpression plants would reduce the hydrogen peroxide scavenging ability of rice.(3)In order to clarify the relationship between rice PKC and rice CatC,we constructed yeast two hybrid vector,pull down vector,Co-IP vector and bimolecular fluorescence complementary(Bi FC)vector of rice PKC gene and CatC gene to verify the interaction between rice PKC protein and CatC protein.The interaction between rice PKC protein and CatC protein was proved from yeast system,in vitro,tobacco system and rice protoplast.(4)In order to further analyze how rice PKC participates in the function o f rice CatC,we obtained three potential PKC phosphorylation CatC sites by ma ss spectrometry,and constructed the point mutation of CatC site to simulate ph osphorylation and dephosphorylation,so as to verify the effect of phosphorylatio n state on CatC.We detected the catalase activity of CatC point mutant proteinand no mutant protein respectively,It was found that the activity of CatC enz yme in simulated dephosphorylation was higher than that in non mutated CatC protein,while the activity of CatC enzyme in simulated phosphorylation was sig nificantly lower than that in non mutated CatC protein.To sum up,this study mainly cloned two rice PKC genes from the rice ge nome,analyzed the expression pattern,and then constructed their transgenic pla nts respectively,and preliminarily identified the high temperature tolerance phen otype of rice PKC,which may be related to rice CatC,so as to verify its inter action with rice CatC and the effect of PKC protein on the enzyme activity of CatC protein,a possible functional model of high temperature tolerance phenoty pe of rice PKC protein was proposed,which provided a theoretical basis and co rresponding experimental materials for further exploring the resistance of rice to stress.