Carrier for constructing hpRNA, constructing method and application thereof

Abstract

The invention discloses a carrier for constructing hpRNA, a constructing method and an application thereof. The DNA fragment sequentially comprises promoter, DNA fragment A, loop nucleotide sequence,DNA fragment B, DNA fragment C and terminator from upper stream to lower stream; the DNA fragment A sequentially consists of LIC process mononucleotide termination site, LIC1 fragment, restriction enzyme A recognition site, fragment formed by the deoxynucleotide which is reversely complemented with LIC3 fragment and LIC process mononucleotide termination site complementation site; the DNA fragmentB sequentially consists of the LIC process mononucleotide termination site, LIC2 fragment and restriction enzyme B recognition site; and the DNA fragment C sequentially consists of restriction enzymeC recognition site, fragment formed by the deoxynucleotide which is reversely complemented with LIC4 fragment and the LIC process mononucleotide termination site complementation site. Only by furthercloning, the carrier for constructing the hpDNA with the DNA fragment greatly improves the constructing efficiency, saves cost, is approximately to be 100% positive, has good performance and stability, and can obtain gene silencing after effective transcription in cisoid expression level and transgene level.

Description

technical field [0001] The invention relates to a carrier for constructing hpRNA, a construction method and application thereof. Background technique [0002] hpRNA will be processed into siRNA in plants to induce RNA interference, and the construction of hpRNA is widely used in RNA interference and gene silencing. The coding region of hpRNA contains two reverse complementary target gene fragments, and a loop region is sandwiched between the two fragments. The coding region is constructed on a plasmid, and a self-complementary single-stranded hpRNA is produced after transcription. [0003] Although the current method of constructing hpRNA has made great progress, there are still defects such as high cost, time-consuming, cumbersome operation, and difficult to master the technology. The hpRNA vectors pHANNNIBAL and pKANNIBAL were constructed by traditional restriction enzymes and ligation systems (Wesley, S., Helliwell, C., Smith, N., Wang, M., Rouse, D., Liu, Q. , Gooding...

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Problems solved by technology

It mainly uses the characteristic of site-specific homologous recombination with the host genome in the lambda phage life cycle, and uses LR reaction to construct (Landy, A. (1989) Dynamic, structural, and regulatory aspects of lambda site-specific recombination.Annu Rev Biochem, 58, 913-949.); Plasmid pHELLSGATE4, pHELLSGATE8 and pHELLSGATE12 all carry out hpRNA construction by this system (Helliwell, C.andWaterhouse, P.(2003) Constructs and methods for high-throughput genesilencing in plants.Methods , 30, 289-295.); However, the construction by the first two plasmids will have unexpected site recombination, resulting in an inverted non-functional intron; while the construction by the pHELLSGATE12 plasmid will ensure that the intron , but the efficiency of its PTGS is unstable (Wesley, S., Helliwell, C., Smith, N., Wang, M., Rouse, D., Liu, Q., Gooding, P., Singh, S ., Abbott, D., Stoutjesdijk, P.et al. (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J, 27, 581-590.); more importantly, in this method The cost of enzymes used is too high

DA-ihpRNA (Xiao, Y., Yin, M., Hou, L. and Pei, Y. (2006) Direct amplification of intron-containing hairpin RNA construct from genomic DNA. Biotechniques, 41, 548, 550, 552.) method It is based on genomic DNA sequence, which uses the intron of a specific gene to generate hpRNA; although this method is simple and fast, the position of the target fragment is greatly limited

Recently, a method for constructing hpRNA in one step has appeared, which utilizes ZeBaTA carrier (Chen, S., Songkumarn, P., Liu, J.andWang, G. (2009) A Versatile Zero Background T-vector System for Gene Cloning and Functional Genomics.Plant Physiol.); In this method, the hpRNA stem-loop structure is obtained by overlapping PCR with two reversed target sequences and any intermediate segment; however, in actual operation, due to the PCR inhibition effect, the One-step generation of hpRNA fragments is difficult to do; moreover, each construct requires the introduction of loop fragments one by one via lap PCR, making this approach difficult as a large-scale screen

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