Cloning And Cold Resistance Functional Verification Of CBF Transcription Factors Gene From Two Kinds Of Ephemeral

Temperature is an important factor limiting the distribution and development of plant.The high temperature influence broader and harm deeper compared with low temperature. The dipartition about types of injury caused by low temperature is complicated.Generally there are freezing, chilling, frost and so on. Many plants gain the capability resisted to freezing by the regulation of CBF gene after short-term and non-lethal low temperature stress.The capability is called Cold acclimation.The cold hardiness research has much room for improvement and feasibility in plant.Traditional plant breeding is generally focused on the drought, salinity and disease resistance and the achievement of traditional cold hardiness breeding in plant is limited.With the rapid development of molecular biology and transgenic technology, the deep research of physiology and biochemistry, signal pathway, especially the research of molecular mechanism of Cold acclimation provide theoretical basis for cold hardiness breeding in plant.The complexity of its geographical environment in Xinjiang leads to bad weather frequently.For example, the temperature has great difference between the day and evening.There are lots of characteristic and biological resources adapted to low temperature stress in Xinjiang. Ephemeral Plants is one of the typical object.These plants germinate in early spring.The snow just melt and the temperature is low at night, sometimes below freezing, frost or even snowing. Its good cold hardiness has attracted attention.Lachnoloma lehmannii Bunge and Malcolmia scorpioides(Bunge) Boiss belong to ephemeral cruciferous plants.CBF transcription factor is an important regulatory factor reply to cold stress by induced the expression of lots cold-related genes.The main results are as follows:1.Explore the optimal conditions for the top expression of the target proteins in prokaryotic expression system.Prokaryotic expression results showed that efficient expression of Ll CBF protein could be realized after induced with 1mmol·L-1 IPTG for 20 h at 30℃ and efficient expression of MsCBF protein could be realized after induced with 2mmol·L-1IPTG for 20 h at 30℃. Solubility analysis showed that the fused protein mainly existed as inclusion bodies.2.The transcriptional activation of the two CBF genes were analyzed by Yeast one-hybrid.The results showed that the transcription factors had transcriptional activation.3.The CBF gene was constructed into the plant expression vector pCAMBIA1304.The recombinant vector was transferred into tobacco by Leaf disc method.Transgenic plants were obtained by PCR verification.The analysis of transgenic tobacco plants reacted to cold by the check of cold-related biochemical indices.The results demonstrated that two kinds of transgenic tobacco surpassed the control in the cold stress.Overexpression of MsCBF genes increased the plant’s tolerance to cold stress proved true preliminarily.