Study On The Regulation Of NRT2.2 Expression By OsCIPK18&Multiplex PCR Detection Method Of Multivalent Bt Gene

It’s all-known that rice is an important food crop,which is applied to gramineous genome and molecular biology research as a model plant.It is great significant to study on its growth and development process.The main nitrogen sources of rice are NO₃^-and NH₄⁺,and 40%of them are absorbed in the form of NO₃^-.In addition to acting as a nutrient element,NO₃^-can also serve as a signal molecule to regulate various stages of botanic growth and development.The signaling mechanism of nitrate signaling in plants involves the CBL-CIPK channel downstream of Ca²⁺signaling pathway.Especially,CIPK is a serine/threonine protein kinase to plants uniquely.However,the literatures related to this pathway have been reported in Arabidopsis thaliana,but little in rice.Previous studies in our laboratory have shown that CBL1and CIPK18 are not only involved in the regulation of rice growth and development,but also participate in the regulation of nitrate signal on rice growth.Previous experiments confirmed the interaction between CBL1 and CIPK18.Apart from this,data of transcriptome indicated that under various nutrient conditions,significantly different expression of genes in two transgenic rice mutants--cbl1、cipk18 were similar.Therefore,CBL1-CIPK18 is likely to regulate rice growth and response to nutritional status on the same signaling pathway.To further elucidate how the CBL1-CIPK18 signaling pathway regulates rice growth and its reaction to nitrogen nutrition,we adopted the following strategies:Firstly,CIPK18 was used as a bait to screen its downstream interaction proteins.More specifically,after constructing the CIPK18-p GBK and CIPK18-p BT3-STE bait vectors,the downstream target proteins were screened and identified by yeast two-hybrid based on the nuclear or DUAL membrane system,to explain the regulation mechanism of CBL1-CIPK18 signaling pathway.Secondly,the promoter of PNR marker gene NRT2.2 controlled by CBL1-CIPK18 pathway was used as a bait,and then the bait vector of p NRT2.2-Ab Ai was constructed.The transcription factors that regulate NRT2.2 expression were expected to find a relationship between NRT2.2transcriptional regulation and CIPK18,and to analyze how the CBL1-CIPK18signaling pathway affects rice perception and responses to nitrate signaling.The following research results were obtained:1.The interaction proteins AMPKβand DUF581 of CIPK18 were screened and identified.AMPKβin yeast is a homologue of Sn RK1 in plants.DUF581 is reported to interact with Sn RK1A in Arabidopsis.These results suggest that CIPK18 may regulate energy state perception in vivo through interaction with Sn RK1.2.HMGB was also screened in the yeast two-hybrid of CIPK18 and the yeast one-hybrid of p NRT2.2.The expression of HMGB was inhibited in both the cbl1 and cipk18 transgenic mutants.These results indicate that CIPK18 may regulate the expression of NRT2.2 by HMGB.3.The CCA1 protein was screened and identified to have an interaction with p NRT2.2 as a bait by the yeast one-hybrid.The expression of the CCA1 gene was also inhibited in both the antisense suppression mutant plants of Os CBL1 and the T-DNA insertion mutant plants of Os CIPK18.In addition,the laboratory has been undertaking the detection of target genes for the insect-resistant transgenic breeding in rice,exploring a simple and effective method for the multivalent Bt insect-resistant transgenic plants.The primers were designed based on the insertion positions of three Bt transgenic events,and used to obtain six target bands with significant differences by multiplex primer PCR technique.Subsequently,the positive and negative bands of the exogenous gene cry1Ab/Ac insertion derived from TT51-1 were 937 bp and 718 bp.The positive and negative bands of the exogenous gene cry2A insertion derived from T2A-1 were 600bp and 434 bp,and the positive and negative bands of the exogenous cry1C insertion derived from T1C-19 were 495 bp and 792 bp.The detection of homozygous,heterozygous and negative Bt transgenic rice plants by agarose gel electrophoresis was rapidly finished.The establishment of this method provides a simple and efficient gene detection technology for insect-resistant rice molecular breeding with multivalent Bt gene,which is beneficial to improve detection efficiency of Bt genes and accelerate the breeding process of insect-resistant transgenic rice.