Space And Temporal Expression Of Alfalfa MsHSP70 And MsTI Gene And Construction Of Alfalfa Somatic Embryogenesis System Mediated Agrobacterium Tumefaciens

Heat shock proteins(HSP) are a class of highly conserved proteins,They are widely distributed in a variety of organisms and play an important role in the biological activities of life.They produce enriched under abiotic stress conditions in plant, and they can reduce the damage produced by abiotic stress. Trypsin inhibitors refer to having trypsin inhibitory polypeptide or protein. they are widely present in a variety of organisms and can adjust many important life events. Trypsin inhibitors are natural insect material and excellent material for plant gene engineering. The objective of this experiment was to study on the role of Ms HSP70 gene in alfalfa fall dormancy and to study the role of Ms TI genes in alfalfa terms of insect resistance, cultivate new insect resistance varieties of alfalfa. The expression of Ms HSP70 and Ms TI gene was studied by quantitative PCR on alfalfa. The gene transformation acceptors selected in this experiment are the Alfalfa cultivar Gongnong NO.1,Xunlu and Vernal.The Ms HSP70 and Ms TI gene,which had been cloned from the Alfalfa cultivar Vernal and ligated to 35 S promoter,was transformed into alfalfa by Agrobacterium-mediated.In the process of transferring alfalfa by leaf disks, we optimized high-frequency regeneration system.Based on that,gene Ms HSP70 and Ms TI,were also transformed into alfalfa by Agrobacterium-mediated. The main results were as follows: Ms HSP70 m RNA showed the relative expression levels increased and then decreased after the first in April, June and September on Stems and buds. It has low expression levels in leaves and increased expression of the roots. In summer, high temperatures can affect the amount of Ms HSP70 m RNA expression, Ms HSP70 overexpression can affect alfalfa fall dormancy.Ms TI m RNA expression relative scale exhibit a decreasing trend in the roots in April and June. The expression of stems, leaves, buds is rise. Establishment and optimization of genetic transformation system by Agrobacterium-mediated. The edges of cotyledons from aseptic plants which had been cultured for 5~7 days were cut, pre-culture for 2 days then moved to a suspension of Agrobacterium cells. Cell density was adjusted to fall 0.5 at OD600. After 15 min inoculation and co-culture 2 days, the explants were transferred to UM1(UM+2,4-D 2.0mg L-1+KT 0.25 mg L-1+CH 2g L-1+ sucrose 30 g L-1+ agar 14 g L-1) containing 500 mg L-1 cefotaxime for 2 weeks.Then, explants were transferred to UM2(UM +KT 1.5mg L-1 +CH 2g L-1+ sucrose 30 g L-1+agar 14 g L-1)for embryo induction and development.Embryos derived from callus were transferred to MS for the formation of roots.