Process of cloning new resistance gene of wild rice

Abstract

The present invention discloses process of cloning new resistance gene of wild rice. The process includes the first constructing library of convertible genomes of donor wild rice, the subsequent designing primer according to the conservation sequences of the cloned rice blast resisting gene and bacterial leaf spot resisting gene to obtain amplified product as the probe, performing bead enrichment on the library with the marked probe to establish candidate resistance gene enriching library, and the final spot hybridization analysis and PCR identification on the cloning of the enriching library, the cloning sequencing analysis and function verification of the amplified product. The present invention integrates candidate resistance gene cloning technology and bead enriching library constructing technology, and is suitable for cloning of wild rice resistance gene and other beneficial plant gene.

Description

Technical field [0001] The invention belongs to the technical field of plant gene cloning, and particularly relates to a method for cloning a new resistance gene of wild rice. Background technique [0002] Wild rice is rich in beneficial genes and is known as the plant giant panda. Due to being in the wild for a long time, wild rice is subject to various pathogenic organisms including viruses, bacteria, molds, and thread during its growth. At the same time, it has to withstand various natural disasters and bad environmental choices. In the course of evolution, resistance to various diseases and insect pests and adaptability to various adversities have been formed, such as disease resistance, insect resistance, cold tolerance, drought tolerance, salt and alkali tolerance, flood tolerance, shade tolerance, and high yield, high quality, male sterility, The high-efficiency utilization of phosphorus and potassium and other traits, these excellent traits are important genetic resources...

AI Technical Summary

Problems solved by technology

Under the condition of simple and stable segregation traits, theoretically any gene can be isolated, but the precondition is that the foundation of preliminary work must be solid, and a fine genetic map including the target gene region must be completed, and a genome library, cDNA library, etc. must be constructed. Libraries must have molecular markers that are very closely linked, and the traits controlled by the target gene must be easy to identify, etc. Since the basic research on the wild rice genome has just started, before the genetic map, physical map, sequence map and expression map are obtained, it is necessary to use This method obviously presents a number of difficulties in isolating specific genes.

[0004] The transposon tagging method uses transposon insertion mutation to inactivate a certain gene, and then uses the transposon-specific gene fragment as a probe to isolate the mutated target gene. This method needs to screen and identify mutants, and the workload is relatively large. And the frequency of effective mutation caused by transposons is very low, about 10 -6 ~10 -4 , but the frequency of transposon cutting from a mutant gene is very high, and the jumping of autonomous transposons is not easy to control. After the transposon is cut off from the insertion site, the original mutation will be restored or new mutations will be generated. The instability of the variation makes it impossible to control and study the bottom frequency variation

Scientists modified the transposon tags to transform them into second-generation transposons—non-autonomous transposon tags, which can stabilize mutations and increase transposition frequency, but double transposon tags still cannot solve transposition The problem of continued transposition of factors

[0005] The isolation and cloning of wild rice new genes by the above cloning techniques requires a thorough study of the target gene and the process is cumbersome and the workload is huge. Therefore, at this stage, it is urgent to find a simple and effective high-throughput technology to isolate and clone new genes, which has important economic value and A new resistance gene in wild rice with broad application prospects

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