| Lysine decarboxylasegene(SaLDC)is a key gene which involved in oxymatrine and matrine biosynthesis and metabolism in Sophora alopecuroides.In this study,Ningxia genuine medicinal materials S.alopecuroides was used as material,based on the existing regeneration system of cotyledon node of S.alopecuroides,the medium was further optimized.Then we use this system and transformed the SaLDC over-expression constructe into cotyledon node of S.alopecuroides by traditional method and in situ transformation method respectively,and got the T0 transgenic plants.And NaCl solution was used to mimic the salt stress.The salt tolerance threshold and physiological characteristics related to salt tolerance during seed germination were analyzed,and the gene expression of SaLDC and the direct product cadaverine(Cad)of the gene were analyzed.Also,we transformed SaLDC over-expression construct into Arabidopsis thaliana by inflorescence infection method.The transgenic Arabidopsis thaliana T2 line was treatment by NaCl solution,which provided theoretical reference for further study on the response mechanism of SaLDC response to salt stress,and laid the foundation for the follow-up gene function research.The main results of this study as follows:(1)The cotyledon node regeneration system of S.alopecuroides was optimized:MS+1.25 mg·L-1 6-BA+0.1 mg·L-1 NAA;The best medium for the proliferation of cluster buds was as follows:MS+1.5 mg·L-1 6-BA+0.1 mg·L-1 NAA+0.3 mg·L-1 IBA;(2)The overexpression SaLDC vector pCAMBIA3301-SaLDC was constructed.The infection solution containing 200μmol·L-1AS,the explants of cotyledon nodes of S.alopecuroides were inoculated with Agrobacterium at the concentration of OD 600=0.5 for 15 min,when Agrobacterium and explant were co-cultured for 1 d,The bacteria were sterilized on:MS+1.25 mg·L-1 6-BA+0.1 mg·L-1 NAA+500 mg·L-1 Cef medium for 6 times,and the highest transformation efficiency was 20.83%.The positive rate of T0 generation identified by PCR was 55.56%,which was an ideal genetic transformation system.(3)Cotyledon nodes of S.alopecuroides seedlings cultured in soil for 7 days were inoculated with Agrobacterium(OD600=0.8)containing over-expression vector pCAMBIA3301-SaLDC for 15 min,the bacteria liquid was co-cultured with cotyledon nodes in dark for 3 days.Cotton balls dipped in 3.34 mg·L-1 6-BA aqueous solution were placed in cotyledonary nodes.After 10 days of light induction under moisture conditions,the rate of regenerated buds was 86.67%.The screening rate of glufosinate resistance was 56.67%and the PCR positive rate was 50.82%.This in situ genetic transformation method effectively improved the genetic transformation efficiency of S.alopecuroides.(4)Salt stress affected the germination and related physiological characteristics of S.alopecuroides seeds.With the increase of NaCl concentration,the seed vigor index decreased and the relative salt damage rate increased.The threshold value of 50%germination rate was 200 mmol·L-1.Under mild salt stress(≤100 mmol·L-1),POD played a major protective role,while CAT played a major role in severe salt stress(>200 mmol·L-1).The expression level of SaLDC in cotyledons was the highest,followed by radicle,and the lowest in hypocotyl;and the gene was up regulated by salt stress;no Cad was detected in cotyledons,but the content of Cad decreased in hypocotyls and radicles with the increase of NaCl solution concentration.There was no significant correlation between the expression of SaLDC and the content of Cad.(5)The transgenic Arabidopsis thaliana seedlings were identified and screened by glufosinate and PCR.When the seeds of T2 generation germinated normally were stressed with NaCl solution for 7 days,the root length of transgenic lines was generally longer than that of wild type,and the number of lateral roots was also more than that of wild type.The effect of NaCl stress on transgenic Arabidopsis thaliana was slightly less than that of wild type.The results of qPCR analysis showed that sa2 was an overexpression plant,and the expression of SaLDC decreased significantly after 7 days of NaCl stress. |
