Physiological Characteristics Of Transgenic Alfalfa And Its Response To Alkali Stress

Alfalfa（Medicago stativa L.）,a perennial legume genus,is one of the most widely cultivated forage crops now.However,the lignin component of alfalfa reduces its digestibility in livestock and affects the economic value.4-coumaric acid coenzyme A ligase（4CL）is a key enzyme of alfalfa in lignin biosynthesis,the lignin content of alfalfa can be reduced effectively by lowering the content of 4CL.The use of antisense RNA technology,putting the antisense 4CL gene of Amorpha fruticosa transferred into alfalfa,successfully acquired transgenic antisense 4CL gene Alfalfa.But when the transgenic alfalfa cultivar is expanded,it faces a serious problem——soil salinization.The problem of soil salinization in China is becoming more and more serious,which has become an important factor restricting the development of agriculture and animal husbandry.The physiological mechanism of transgenic alfalfa in response to soil alkali stress has no report so far.This experiment material uses alfalfa which has turned 4CL antisense gene with low lignin and study its physiological characteristics,then study the effects of soil alkali stress on physiological and biochemical and gene expression of T1 generation transgenic alfalfa.The research made the following progress:1)T1 generation alfalfa were identified as positive transgenic alfalfa by PCR detection,which indicated that antisense 4CL gene had been inherited to the next generation and could be expressed normally.The results of enzymatic activity and lignin content determination showed that the introduction of antisense 4CL gene could effectively reduce the synthesis of 4CL enzyme,and the lignin content of T0generation alfalfa was 54.44%lower than that of wild type alfalfa.The result of net photosynthetic rate of transgenic alfalfa in T0 generation and T1 generation showed that the net photosynthetic rate of transgenic alfalfa was higher than that of wild type under the same light intensity.Experimental results show that the introduction of antisense 4CL gene can effectively reduce the content of alfalfa lignin at the same time it will not affect the normal growth of alfalfa,Under the same light intensity,the net photosynthetic rate of alfalfa with antisense 4CL gene was higher than that of wild type alfalfa..2)The alkali-resistance physiology of wild type and T1 generation alfalfa was studied in this paper,which include the content of perhydrol（H₂O₂）、superoxide radical（O₂^-?）,activities of catalase（CAT）、peroxidase（POD）、superoxide dismutase（SOD）,content of malonaldehyde（MDA）,proline,chlorophyll and relative conductivity of leaves.The results showed that transgenic alfalfa had low content of H₂O₂、O₂^-?,and there was no significant change in MDA content and leaf relative conductivity of transgenic alfalfa compared with wild alfalfa,and no significant difference in antioxidant enzyme activity between transgenic alfalfa with the increase of alkali concentration.This indicates that transgenic alfalfa is less sensitive to the change of external environment,less damaged by alkali stress,and more resistant to alkalinity.3)The gene expression of antioxidant enzymes in wild type and T1 generation alfalfa was determined under alkali stress.The results showed that the expression of three types of SOD and CAT genes in wild type alfalfa showed a strong stress with the increase of alkali concentration,and no significant difference in POD gene expression among different concentration groups.However,the trend of expression of Mn-SOD、Fe-SOD and CAT genes in transgenic alfalfa was gentle,no significant difference between different treatment concentration groups.But Cu/Zn-SOD and POD gene showed high expression level.In general,the gene expression of wild alfalfa was higher than that of transgenic alfalfa.The amount of gene expression was the molecular compensation to enzyme activity.The gene expression of wild type alfalfa showed high stress,but the general change trend of transgenic alfalfa gene was not obvious.The molecular level showed that transgenic alfalfa had low sensitivity and good tolerance.