Zizania latifolia complementary deoxyribonucleic acid (cDNA) library construction method

Abstract

The invention relates to the field of molecular biology, and in particular discloses a zizania latifolia complementary deoxyribonucleic acid (cDNA) library construction method. The zizania latifolia cDNA library construction method provided by the invention takes tender leaves of zizania latifolia as a test material, and comprises the twelve steps of ribonucleic acid (RNA) extraction, cDNA first strand synthesis, long-distance polymerase chain reaction (LD PCR), proteinase K digestion, cDNA library homogenization, twice PCR amplification, amplified product purification, Sfi I digestion, enzyme digestion cDNA purification, connection of cDNA and a PUC19 carrier, transformation of plasmid, cloning identification, and the like. The method is convenient and fast in library construction; the constructed library is high in capacity, high in recombination rate and good in integrity; after homogenization, high-abundance genes are obviously reduced, so that a foundation is laid for the verification of functional genes. The zizania latifolia cDNA library constructed by the method is high in capacity, and the capacity reaches 3.6*10<6>pfu.mL<-1>; the average length of an inserted fragment is 1000bp, and the recombination rate is 97.9%; the normalized full-length cDNA library can effectively enrich low-abundance expression genes, thus reducing a redundancy rate. The zizania latifolia cDNA library is used for screening a target gene and for subsequent gene and protein interaction analysis.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a method for constructing a wild rice cDNA library. Background technique [0002] With the rapid development of molecular biology and bioinformatics technology, mining, cloning new genes, and studying the functions of new genes have become an important task in functional genomics research. The construction of cDNA library is one of the basic means of functional genomics research. It has special advantages in the study of the expression state of the genome in a certain type of cell and the functional identification of expressed genes. It has a wider application value in the study of life phenomena such as apoptosis and apoptosis. However, the biggest problem faced by cDNA libraries constructed by conventional methods is the high proportion of duplicate copies in the process of high-throughput analysis. The main reason is that the gene expression levels in cells of higher eukaryot...

AI Technical Summary

Problems solved by technology

However, the molecular biology research on wild rice is still in its infancy, and the genetic information of wild rice is still incomplete. The limited knowledge of wild rice genetic information seriously hinders its research and application as an excellent gene resource bank for rice breeding.

Therefore, a fast, simple and efficient method for constructing wild rice cDNA library was developed to solve the problems of complex routine operation steps and easy degradation of RNA, overcome the huge difference in gene transcription level, and increase the probability of discovering random sequences and rare genes. It is very necessary to study the utilization of functional genes in wild rice

Images