Construction Of A CDNA Library From Root Tissue And Screening Of Functional Genes Of Dongxiang Wild Rice

Rice is an important crop that production wes related with security and stability in the national economy. The yield of rice wes reduced due to the cold stress and other abiotic stresses in every year. To improve rice tolerance to these abiotic stresses wes reduced some stress by genetic improvement, and it ensuring high and stable yield in rice.Dongxiang Wild Rice (Oryza rufipogon) is the northernmost wild rice in the world known to date and has extremely high cold tolerance and many other adversity-resistant properties. In order to identify the genes involved in cold tolerance and other signal pathways in Dongxiang wild rice, In this study, The cDNA library was constructed by SMART technology with root tissue of Dongxiang Wild Rice in winter.First, total RNA was prepared from the root tissue of Dongxiang wild rice, the first and second cDNA strands were synthesized by SMART method and LD-PCR amplification, respectively. Second, different cDNA size fractionation using CHROMA SPIN Columns and collecting larger cDNA fragment. Then, the double strand cDNA (ds cDNA) fragments were ligated to λ TriplEx2Vector and packaged in Lambda DNA. Finally, the recombinant X TriplEx2vector wes convert to pTriplEx by transformation into E.coli, and the plasmid cDNA library of Dongxiang wild rice was successfully constructed. The results showed that titer of the unamplified cDNA library was2.9×106cfu/mL, the average length of inserts about800bp and the recombination rate reached to99.5%.To identify functional genes involved in stress tolerance and other metabolic pathways, more than1000clones from cDNA library were sequenced. Functional genes including Zinc finger protein, Probenazole-inducible protein, abscisic acid-inducible protein, Metallothionein-like protein, Proteinase inhibitor were characterized. By bioinformatical analysis, the No.155clone with a putative open reading frame (ORF) of567bp was predictably encoded a Zinc finger protein of188amino acid (aa) with a molecular weight of24.4kD that functioned as a transcription factor, and structure of this protein was analysized. The PCR product with template of No.155clone was ligased into pET-28a, and the recombinant vector of pET-28a-155were successfully constructed by sequencing identification.To elucidating mechanism for the signal transduction in response to low temperature stress of Dongxiang wild rice, and the primers were designed for according to the Nipponbare’s CBF-3gene sequence. The CBF-3gene of Dongxiang wild rice wes amplification by PCR. And the results show that the same sequence between Dongxiang wild rice with the Nipponbare.The cDNA library with root of Dongxiang wild rice wes constructed, and many gene incloud to stress tolerance gene and other function gene were screened out.Then, the pET-28a-155prokaryotic expression vector wes successfully constructed, and the CBF-3gene of Dongxiang wild rice wes cloned. This study will provide foundational basis for cloning and characterization of favorable genes in Dongxiang wild rice.