Studies On Genetic Transformation And Droung-Resistance Of Alfalfa HD-Zip Transcription Factor Gene Mfhb-1 In Tobacco And Wheat

Alfalfa Mfhb-1 gene is at the beginning of the alfalfa somatic embryogenesis,obtained by excluding hybrid type belongs to the homologous exotic leucine zipper proteins（Homeodomain-leuzine zipper protein,HD-Zip）Ⅰclass transcription factor genes.At present,the most common Transcription factor is a specific protein that can combine with some cis-acting elements of genes to make biological regulation on plants at the Transcription level.Previous studies have shown that homologous-leucine zipper（HD-zip）protein is a class of transcription factors unique to higher plants,which are mainly involved in plant physiological processes such as response to stress,regulation of embryonic development,and photomorphogenesis.In order to clarify the biological regulation function of Mfhb-1 gene under plant stress,we transfected the alfalfa Mfhb-1 gene into tobacco and wheat by Agrobacterium-mediated method,respectively,and identified the transgenic tobacco and wheat by molecular biology.The activities of SOD and POD,the content of MDA and the content of chlorophyll in tobacco were measured under drought stress,and the biological regulation function of Mfhb-1 gene was determined.The main research results were as follows:（1）Genetic transformation of Mfhb-1 gene to tobacco was completed by Agrobacterium tumefaciens mediated method.Transgenic tobacco plants were obtained through infection,co-culture,debacteriosis,screening and other processes,and the number of resistant callus in the process of genetic transformation was counted.The optimal conditions of Agrobacterium mediated tobacco genetic transformation were determined as follows:the OD₆₀₀ value of infection solution was 0.61,and the infection time was 20 min.（2）Ten homozygous transgenic tobacco lines with Mfhb-1 gene in T₃ generation were obtained by the combination of HYG spray and PCR detection.（3）RNA was extracted from the T₃ generation of homozygous tobacco and reverse transcription was carried out.The c DNA generated by reverse transcription was detected by RT-PCR.The results showed that both transgenic tobacco plants and positive plasmid DNA could amplify the target band with a length of 200 bp,indicating that Mfhb-1 gene was expressed in tobacco.（4）Firstly,the tobacco plants successfully transferred into alfalfa Mfhb-1 gene were subjected to drought stress for different time,and then the tobacco plants under drought stress were observed and a number of physiological indexes were measured.The results showed that,（1）under the condition of normal growth,no difference between the two types of the apparent form of tobacco,with continuous drought stress time,wild type tobacco plants present a greater negative impact of change,Including yellowing of leaves,changes in leaf crimp,and drying up of stems,the apparent morphological changes of Mfhb-1 transgenic tobacco plants were similar to wild-type tobacco plants,but the degree of change was far less than that of wild-type tobacco plants.Under the condition of drought stress for 20 days,it still showed a good growth trend.（2）SOD activity of Mfhb-1 transgenic tobacco was significantly higher than that of wild-type tobacco under both normal and drought stress,The highest activity was observed under drought stress for 20 days;under normal growth conditions,there was no significant difference in POD activity between the two tobacco types.However,under drought stress,the POD activity of transgenic tobacco increased significantly more than that of wild-type tobacco,and the POD activity increased by 37.11%at 20 days drought stress.（3）There was no significant difference in water content between wild-type and Mfhb-1 transgenic tobacco plants under normal growth conditions,both of which were more than 80%;Under drought stress for 15 days,the water content of both types of tobacco decreased,but there was no significant difference in water content between them at this time;After20 days of drought stress,the water content of both plants continued to decrease,and the water content of transgenic tobacco was significantly higher than that of NC89 wild-type tobacco;In contrast to tobacco moisture content,MDA content of the two types of tobacco showed no significant difference under normal growth conditions and remained at the same level,However,MDA content of transgenic tobacco was significantly lowerr than that of wild-type tobacco when drought stress lasted for 15 days.（4）The content of soluble sugar and Pro in transgenic tobacco was not significantly different from that of wild-type tobacco under normal growth conditions,but the content of soluble protein was significantly higher than that of wild-type tobacco;Under drought stress for 15 days,the contents of Pro,soluble sugar and soluble protein of Mfhb-1 transgenic tobacco were significantly higher than those of wild-type tobacco,and the difference of Pro content was the largest.Under drought stress for 20 days,the soluble protein and soluble sugar contents of transgenic tobacco were significantly higher than those of NC89 wild-type tobacco,but there was no significant difference in Pro content between the two tobacco.Under normal growth conditions,there was no significant difference in chlorophyll content between the two types of tobacco,and both of them were at a normal level;The chlorophyll content of transgenic tobacco was significantly higher than that of NC89 wild-type tobacco when the drought stress time reached 15 days;The chlorophyll content of transgenic tobacco was higher than that of wild-type tobacco with prolonged drought stress.These results show that in the absence of stress conditions,Mfhb-1 transcription factors will not affect the growth of plant physiology,exogenous drought stress,Mfhb-1 transcription factors can improve the plant protection enzymes activity,at the same time in the form of increasing osmoregulation substance content to enhance cell osmotic potential to improve water imbibition,slowing plants to reduce moisture content,suggests Mfhb-1 gene expression may be induced by exogenous drought stress condition.（5）By agrobacterium agrobacterium mediated method and the vacuum method for wheat genetic Mfhb-1 in the form of transformation,during the period of germination and growth of wheat to make use of hygromycin preliminary screening,preliminary screening for Hyg resistant wheat plant,PCR tests again to screen out wheat,AGAR gel electrophoresis results showed that transgenic wheat plant and e.coli plasmid DNA in 1300 bp in the length of clear stripe,can further confirm Mfhb-1 gene has been successfully transferred into wheat.（6）The number of wheat plants identified as positive by PCR was counted.Combined with the different conditions of genetic transformation of wheat,the highest success rate of genetic transformation of wheat was obtained,the OD₆₀₀ value of infection solution was 0.41,and the success rate of transformation of wheat was 2.4%when the duration of vacuum state was 30 s.in conclusion,this study preliminarily clarified the drought-resistant function of Mfhb-1 gene,through multiple physiological indexes of transgenic tobacco found that compared with wild type tobacco plants,import Mfhb-1gene of tobacco in the normal and at different time under the condition of drought stress,SOD and POD activity,MDA content,Pro content and other physiological indexes have varying degrees of change,all point to the changes of drought resistance,in order to further research the HD-Zip function of transcription factors and mechanism provides a theoretical basis.In addition,alfalfa Mfhb-1gene was successfully introduced into wheat by Agrobacterium tumefaciens mediated method assisted by vacuum extraction,which provided a reference for the further study of monocotyledonous transgenic technology.