Study On Differential Gene Function In Regeneration Of Alfalfa With Different Fall Dormancy Levels

Medicago sativa L.is a feed crop with high economic value and rich in protein,and it is an important leguminous forage in the production of agriculture,animal husbandry and aquaculture.At the same time,as a high-quality plant protein source,it is the support for the development of dairy industry and is of great significance to the development of animal husbandry.Alfalfa stems and leaves have high nutritional content and are the main components of animal feed.Because of cross pollination of alfalfa,most varieties have complex genetic background.Limited by genetic characteristics,its growth performance and nutritional quality are uneven.Alfalfa stems are composed of nodes and internodes,which affect plant height and yield.The above ground parts（height,stem diameter and number of branches）are important factors limiting its crop yield.It is very important to increase the number of alfalfa vegetative branches,vegetative growth time and delay the flowering time of plants.Objective:The purpose of this study is to screen out the significant genes related to alfalfa growth from alfalfa varieties with significant differences in growth performance.The expression vector was constructed,and the wild Arabidopsis thaliana was transformed by Agrobacterium-mediated method.Through the detection of agronomic characters,molecular and transcription expression levels,the functions of growth-related genes were preliminarily explored,which laid the foundation for exploring the signal pathway that they might participate in the vigorous growth of alfalfa,provided reference for molecular breeding of alfalfa,and opened up a new way for the high-quality development of animal husbandry.Method:Experiment 1:The growth performance and nutritional quality of 10 alfalfa varieties with different fall dormancy levels were measured and analyzed at the initial flowering stage.Experiment 2:Determine two alfalfa varieties with significant differences in growth performance and their rapid growth period.In order to ensure the sufficiency of experimental materials and the consistency of genotypes,the cutting propagation of alfalfa with significant growth differences and representative varieties was carried out.The stem tissue was collected for transcription sequencing and conducting biological informatics analysis,and preliminary identification the differential genes related to rapid growth and the related metabolic pathways involved.Experiment 3:Two target genes（auxin inducible protein gene ARG4 and chlorophyll ab binding protein gene CAB6）with significant differences related to rapid growth were screened out,and the expression vectors were cloned and constructed,and the agrobacterium tumefaciens was transformed.Experiment 4:Agrobacterium tumefaciens-impregnated inflorescence method mediated genetic transformation of Arabidopsis thaliana plants,and cultivated to maturity to obtain T₀ generation seeds;The positive seedlings were preliminarily screened by antibiotic screening medium,and after molecular biological identification,they were cultivated to maturity to obtain T₁ seeds.According to this method,T₃seeds are finally harvested.Experiment 5:The molecular identification of T₄ generation positive seedlings was carried out,the growth index and the transcriptional expression level of the target gene were determined,and the function of the target gene in plant growth was preliminarily confirmed.Results:Experiment 1:The growth index（plant height,stem diameter,branch number,internode length,yield per plant,etc.）,nutritional index（crude protein,neutral detergent fiber,acid detergent fiber,dry matter,etc.）and overwintering rate of 10 different fall dormancy levels of alfalfa were analyzed and compared based on the membership function and cluster analysis.It was found that the production performance and nutritional quality of（extreme）non-fall dormancy alfalfa groups represented by"Yulu 1,WL712"were better;The autumn dormant varieties represented by"Aohan and Zhaodong"have poor comprehensive performance.Experiment 2:Through dynamic analysis of different varieties,the fast-growing"WL 712"and the slow-growing"Aohan"were taken as the research objects.Transcriptome sequencing analysis was carried out on the middle stem tissues of two varieties in bud stage.KEGG analysis showed that plant hormone signal transduction,phenylpropanol biosynthesis and photosynthesis genes were highly expressed in"WL712".GO analysis found seven DEGs clusters related to the biological process of plant growth and development:vascular tissue formation,cell division and bud formation,lignin synthesis and degradation,stem growth,primary or secondary cell wall formation,cell enlargement and plant growth,induced germination and cell elongation.ARG4 gene involved in cell initiation and plant growth biological process and CAB6 gene encoding chlorophyll a-b binding protein,which is involved in regulating plant Senescence pathway and highly expressed,were preliminarily used as growth-related candidate genes for functional verification.Experiment 3:The auxin inducing protein gene（ARG4）and chlorophyll ab binding protein gene（CAB6）of alfalfa were cloned,and the plant expression vectors p BI121-ARG4 and PCAMBIA1300e GFP-CAB6 were successfully constructed.Experiment 4:Using the genomic DNA of the T₁ generation p BI121-ARG4 or PCAMBIA300e GFP-CAB6positive plants as templates,561 bp or 720 bp electrophoresis bands were obtained by PCR amplification.In order to further verify the accuracy of PCR,c DNA was used as a template for full-length amplification,and the electrophoresis results were consistent.It was preliminarily believed that the target gene At ARG4 or At CAB6 had been successfully integrated into the Arabidopsis genome.Experiment 5:（1）The results showed that the 1000-grain weight of T₃ generation seeds of p BI121-ARG4and PCAMBIA1300e GFP-CAB6 positive seedlings were significantly higher than that of the wild type and negative control,while the germination rate was opposite.The young root length,plant height,pod length and the number of seeds per pod of T₄ seedlings were significantly higher than those of wild-type and negative control plants.The plant height and branch number of the positive plants at different cultivation times were significantly higher than those of the wild-type and negative control,and the transcription and expression level of At ARG4/At CAB6 gene in the stem and leaf tissues were significantly higher than those of the wild-type and negative control.（2）When the light duration was shortened,the number of rosette leaves of PCAMBIA1300e GFP-CAB6positive plants was significantly the largest,and the number of branches and plant height were significantly lower than those of wild type and negative control.The transcription and expression level of At CAB6 in leaf tissue was significantly higher than that of other plants,and there was no significant difference in the transcription and expression in stem tissue.We speculate that the decrease of light time makes At CAB6 gene more likely to be transcribed and expressed in rosette leaves.Conclusions:954 DEGs related to rapid stem growth in the stem tissue of vigorous growing"WL 712"and slow-growing"Aohan"alfalfa,which are mainly involved in the formation of vascular tissue,cell division and bud formation,lignin synthesis and degradation,stem growth,primary or secondary cell wall formation,cell enlargement and plant growth,and induced germination and cell elongation and other biological processes.Genes related to plant hormone signal transduction,phenylpropane biosynthesis and photosynthesis are highly expressed in"WL 712".It was preliminarily found that the plant height,number of branches,weight of 1000 seeds,length of pods,and number of seeds in a single pod of At ARG4/At CAB6transformed plants had significant changes.In addition,the shortening of illumination time led to a significant increase in the number of rosette leaves in At CAB6 transformed plants.In this study,the genes related to growth regulation were excavated from alfalfa,and the over-expression materials of growth-related gene ARG4/CAB6 were created,which provided theoretical basis for molecular breeding of alfalfa.