Study On Transformation Of Citrate Synthase Gene In Medicago Sativa L.

In recent years,with the improvement of living standards,the demand of people for livestock products was also increasing;In animal husbandry,the forage and feed were the foundation,in North China,the quality of forage was high,but the yield was low because of drought,which restricted the development of animal husbandry in North China,in South China,the condition of water and heat were good,the yield of forage was high,but its quality was not good,In general,the demand of current market on animal husbandry cannot be meted because of the lack on the forage and feed in our country.Especially,a major obstacle on animal husbandry development in South China was the lack of high quality leguminous forage.Alfalfa was recognized as the high quality leguminous forage in the world,if it can be introduced to the south of China,there will be a large urge action on the development of animal husbandry.But the soil was acid in South China,and the aluminum toxicity on the acid soil was adverse on alfalfa growth especially,which limit the alfalfa southwards greatly and affect the development of alfalfa industry in South China seriously.Thus,the cultivation of new alfalfa varieties which have tolerance to aluminum toxicity was the key to solving the alfalfa southward.In this study,the alfalfa variety named "Yu Mu No.1" was used as the material,we transformed light-induced and constitutive promoter CS into alfalfa genome using Agrobacterium-mediated leaf disk method.And then the transgenic plants with CS gene were identified by PCR,RT-PCR and real-time Q-PCR.CS enzyme activity in leaves,the citrate content in leaves and root tips and citrate secretion in the transgenic plants were tested,their abilities to tolerate aluminum were measured,and the differences between different promoters drived gene in the receptor plants were compared.The major results in this study were as follows:1 Thirty-three plants with resistance were obtained by transgenic operation,in which fifteen were light-induced and eighteen were constitutive,six positive transgenic plants were detected by PCR identification,in which CS-2,CS-3,CS-18,CS-66 were constitutive and rCS-4,rCS-9 were light-induced;Five of the six transgenic plants can transcribe in RNA level by RT-PCR identification,in which light-induced promoter transgenic plants were rCS-4?rCS-9,constitutive promoter transgenic plants were CS-2,CS-3 and CS-18,In which the CS copy multiple in the genome of CS-2,CS-3,rCS-4,rCS-9 compared to WT were 701.03,1076.15,10.69 and 107.39.2 CS enzyme activities of transgenic plants:CS enzyme activities of transgenic plants were higher than the wild type plant and their difference reached extremely significant level.The value of CS enzyme activity of constitutive promoter transgenic plant CS-2 was 0.09 NADH/min/mg protein,which was the highest of all and 1.26 fold of the wild type plant,the second was constitutive promoter transgenic plant CS-3,its value was 0.086 NADH/min/mg protein.CS enzyme activities of transgenic plants CS-3?CS-18?CS-66 were higher than the light-induced promoter transgenic plant rCS-4 and their differences reached extremely significant level,but compared to light-induced promoter transgenic plant rCS-9,there were no significant difference.3 The content of citric acid in leaves and root tips of transgenic plants:The content of citric acid in leaves and root tips of transgenic plants were higher than the wild type plant;the content of citric acid in leaves of constitutive promoter transgenic plant CS-3 was 86.74?mol/g fresh leaf,which was the highest of all and 1.56 fold of the wild type,the followed were CS-2 and rCS-9,their values were 78.24 and 78.12?mol/g fresh leaf respectively;the content of citric acid in leaves of light-induced promoter transgenic plant rCS-4 was the lowest;the content of citric acid in root tips of constitutive promoter transgenic plant CS-3 was 24.01?mol/g fresh root tips,which was the highest and 2.05 fold of the wild type,the followed was CS-3,its value was 19.57?mol/g fresh root tips;the difference between different promoters transgenic plants reached significant level,thus the content of citric acid in root tips of constitutive promoter transgenic plants were significantly higher than the light-induced promoter transgenic plants.4 The citric acid secretion in root system of transgenic plants:the values of citric acid secretion of transgenic plants and the wild type were all low,the citric acid secretion of the wild type and the most transgenic plants were equal under no aluminum stress;when under 20?M aluminum stress,the citric acid secretion of each plants obviously increased,and the values of citric acid secretion of transgenic plants were all higher than the wild type,the values of CS-2 and CS-3 were the highest,which were 0.371 and 0.368?mol/l,2.22 and 2.20 fold of the wild type respectively.5 The root elongation and the staining of root tips of transgenic plants:under no aluminum stress,all the plants growed nomally,the values between the wild type plant and the most transgenic plants were equal,and there were no staining in all root tips.under 20 ? M aluminum stress,the root growth of all plants were inhibited,the root elongation of transgenic plants were longer than the wild type plant,and the differences between them reached extremely significant,the root elongation of CS-2 up to 0.67cm was the longest,and 2.98 fold of the wild type.There were different degrees staining in different plants,the stained area of the wild type was the largest,and its staining was also the deepest of all the plants.The conclusions of this study were following:The CS gene could be integrated into the genome of all the transgenic plants,it could effectively improve the CS enzyme activity of transgenic plants and increase the citric acid content in leaves and root tips of transgenic plants.Thus,when under aluminum stress,the transgenic plants could secrete more citric acid to resistance the aluminum toxicity,and show stronger aluminum tolerance.Constitutive promoter compared to light-induced promoter,it was more conducive to the expression of CS gene.