Cloning And Analysis Of CBF Gene From Lachnoloma Lehmannii And Malcolmia Scorpioides

Low temperature adversity stress is a key factor confining plant growth. When a plant is under adversity stress, a series of stress resistance gene expressions will be activated. Early-spring ephemeral plants are a typical group of plants that completes their life in a quite short period of time during spring or summer. Since they can grow under low temperature, it is safe to infer that they have effective low temperature resistance mechanism. Study is done on low temperature resistance evaluation, CBF genes clone related to low temperature resistance, bioinformatic analysis, expression level analysis and CBF gene prokaryotic expression vector construction with three kinds of early-spring ephemeral plants, trying to lay a foundation for further research on gene and provide evidence for in-depth study of low temperature resistance mechanism of early-spring ephemeral plants.1. Three kinds of early-spring ephemeral plants are put under4℃environment to undergo adversity stress. Seven physiological and biochemical indices are tested to generally evaluate their ability in resisting low temperature. Results show that the order is Malcolmia scorpioides (Bunge)Boiss> Lachnohma lehmannii Bunge> Alyssum dasycarpum Steph. ex Willd. A single biochemical index can hardly reflect plants’ ability in resisting low temperature, while relative conductivity, free proline content, soluble sugar content, SOD and POD enzymic activity can better reflect plants’low temperature resistance ability and can act as reference indices for evaluating low temperature resistance ability of spring ephemeral plants.2. CBF genes of Lachnoloma lehmannii and Malcolmia scorpioides are cloned with homologous cloning strategy and RT-PCR technology, and verified with full-length primer design. The sequencing results show that CBF genes of Lachnoloma lehmannii and Malcolmia scorpioides are successfully obtained. The genes’sequence are submitted to GenBank database and accessing numbers of Lachnoloma lehmannii and Malcolmia scorpioides are obtained as JQ687132and JQ687138, respectively.3. Structure and function of CBF genes from Lachnoloma lehmannii and Malcolmia scorpioides are analyzed with bioinformatic method. The CBF genes of Lachnoloma lehmannii and Malcolmia scorpioides as well as their deduced proteins share the same feature of CBF gene family. It is initially inferred that CBF genes of Lachnoloma lehmannii and Malcolmia scorpioides have the same function as CBF genes of other plants. CBF gene of Lachnoloma lehmannii is named as LICBF gene and Malcolmia scorpioides MsCBF gene.4. Fluorescent quantitative RT-PCR technology is adopted to analyze low temperature induction expressions levels of LICBF and MsCBF with Actin gene as the reference. The results show that after2hours of stress, the expression level of MsCBF reaches maximum being10times higher than the reference. After6hours of low temperature stress, the expression levels of LICBF gene in root, stem and leaves reached maximum, and after24hours, dropped to level equal to the reference. Results indicate existence of low temperature induction of the gene’s expression which is related to low temperature resistance of Lachnoloma lehmannii and Malcolmia scorpioides.5. MsCBF expression primers with digestion site are designed and ORF of MsCBF gene with two enzymes digestion sites on both ends is expanded. This segment is inserted to prokaryotic expression vector pET-28a with DNA recombination technology. CBF prokaryotic expression vector pET-28a-MsCBF is thus successfully constructed.