Cloning And Functional Verification Of Inducible Promoter Of Amygdalus Ledebouriana Schlecht

At present,the cold resistance and drought resistance of Xinjiang wild almond(Amygdalus Ledebouriana Schlecht)have been studied mainly from the biological characteristics,physiological and biochemical detection,resource distribution and other aspects,seldom from the perspective of gene regulation expression.In the regulation of gene expression,inducible promoter can induce the expression of related genes response to the change of external environment,according the needs of plant in growth environment,to reduce the waste of plant nutrition.Therefore,the study of promoter function and mechanism will be conducive to the understanding of gene expression and signal transduction pathway.The promoter of CBF1 gene was cloned from Xinjang wild almond,and the function of the promoter was verifies,the major results were as follows:1.CBF1 promoter of 1791 bp have been cloned from wild almond(GenBank accession number KX785336),and named AlsCBF1 pro.Bioinformatics analysis showed that the sequence contains 20 low temperature stress elements MYCCONSENSUSAT,and a large number of elements could respond to salt,hormone,drought and other abiotic stress.2.The plant expression vector of CBF1 gene was constructed,and transformed in tobacco by leaf disc transformation.The transgenic plants were obtained and stained with GUS after being stressed by low temperature,drought,ABA and salt,and the result showed that the promoter could drive the expression of GUS gene under low temperature,drought,ABA and salt stress.3.The plant expression vectors of six AlsCBF1 pro 5? tuncated fragments were constructed and transformed in tobacco,then analyzed through GUS fluorescence quantitative analysis after being stressed by low temperature,drought,ABA and salt stress,showing that the expression of full-length and truncated fragments were different.The sensitivity of full-length to stress was low,while 5? truncated fragments Q2,Q4,Q5,Q6 were high,especially in response to low temperature stress,elements of MYCCONSENSUSAT between AlsCBF1pro-1076~-1339 bp and-30~-380 bp play a major role in low temperature stress;elements of MYB and MYC between-22~-768 bp are related to drought stress;there may be some elements affecting the promoter to regulate salt stress between-1339~-1791 bp,and the deletion of-1~-1076 bp could affect the regulation and expression of promoter on ABA stress.