Construction Of The RNAi Vector Of Ramie CCoAOMT Gene And Its Transformation In Tobacco

Alfalfa(Medicago sativa L.)is a perennial legume with the advantages of applicability,high yield and good quality,which can improve the soil and conserve both soil and moisture.Although the resources of Alfalfa Varieties in China is very rich,but its digestibility is low.The primary task of the breeders is to improve the digestibility of alfalfa and improved its quality.The studies in improving the digestibility showed that the digestibility of alfalfa dry matter has significant negative correlation with the content of lignin.So a practical way to improve the digestibility and changthe nutritional characteristics is to regulate the biosynthesis pathway of lignin scientifically and reduce the lignin content of alfalfa to improve digestibility.Lignin is one component of plant cell walls and wildly exists in plants,which is mainly located between cellulose fibers,with the role of compression,supports the plant and resist pests and diseases.But the lignin can not be digested by domestic animal and its economic value of forage plants is seriously affected,so it is particularly important to reduce the plant lignin content by genetic engineering methods.Coffee acyl coenzyme A-O-methyltransferase(CCoAOMT)is a key enzyme in lignin biosynthesis pathway.By inhibiting of CCoAOMT activity,it can reduce the synthesis of lignin effectively,which can reduce the content of plant lignin.In order to achieve the purpose of reducing alfalfa cell wall lignin biosynthesis,we completed a basic work in the following two aspects in this study.Firstly,we constructed CCoAOMT gene RNAi expression vector,and verify the biological function of the vector by transforming the model plant tobacco;secondly,the establishment of efficient regeneration system of alfalfa cotyledon,lay the foundation for the genetic improvement of alfalfa.This experiment propagated two highly conserved sequence of CCoAOMT gene in alfalfa,and the amplified fragments were connected to both sides of the CSHA intron of plant interference expression vector pFGC5941,constructed a plants interference expression vector pFGC5941-CCoAOMTi.By putting the vector into tobacco by Agrobacterum-mediated to obtain transgenic plant.By PCR analysing and the determination of plant lignin content,getting the first generation of transggenic plants,and the positive plants were survivalled,blossomed,got the seeds.Through the analysis of PCR and RT-PCR to the second generation,the interference gene was integrated into the transgenic plants and expressed.The detection of physiological and biochemical indexes of transgenic plants showed that the lignin content was reduced and maintained at a suitable range,and the plants grew normally.The results indicated that alfalfa CCoAOMT gene expression vector which we were constructed could effectively inhibit the expression of the corresponding gene synthesis of tobacco,and reduce the synthesis of the desired product.It is reasoned tha the expression of the carrier in alfalfa may have more significant biological effects.This study used cotyledonof "Gannong 1" alfalfa as explant,and this study successfully constructed a high-frequency regeneration system of alfalfa by adjusting the basic medium and the concentration of various hormones.In this system,the alfalfa callus induction rate was 95%,the embryoid induction rate was 72%,and the rooting rate can reach 100%,the system had prayed the way for the alfalfa as transgenic receptor.This study lays a foundation for the genetically engineered alfalfa.