Molecular identification method of HMGR gene mRNA in pear stock-scion transfer

Abstract

The invention discloses a molecular identification method of HMGR gene mRNA in pear stock-scion transfer. The molecular identification method comprises the following steps: (1) by taking pyrus bretschneideri rehd as scion and pyrus betulifolia bunge as stock, carrying out micro grafting of test-tube seedlings; (2) extracting total RNA of pyrus bretschneideri rehd, pyrus betulifolia bunge and grafted stock and scion, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification on the total length of the HMGR gene, and carrying out second-time PCR by adopting the amplification result as a template, thus obtaining respective HMGR gene fragments, wherein the sequences of upstream and downstream primers obtained through two times of PCR are respectively shown as SEQ ID NO.1-4; and (3) carrying out enzyme digestion on the PCR product obtained for the second time by adopting restriction endonuclease, and comparing enzyme digestion spectrograms before and after grafting for identifying the transfer condition. The method provided by the invention is rapid and sensitive, is high in accuracy, is simple and convenient, and can be applied to other pear plants.

Description

technical field [0001] The invention relates to the field of plant molecular biology, in particular to a molecular identification method for the long-distance transfer of HMGR gene mRNA molecules between rootstocks and scions of the genus Pyrus. Background technique [0002] In the practice of horticultural crop production, the application of grafting technology can improve plant resistance, increase yield, and improve fruit quality. used widely. In the production of fruit trees, it is found that different rootstocks can have different effects on the physiological and biochemical characteristics of the scion, flowering and fruiting, environmental adaptability, and tree growth and development, and these effects will eventually affect the formation of fruit and the later economic value. to a crucial role. [0003] So far, it has been found that there are many endogenous mRNA molecules that are transmitted long-distance through the plant phloem between the rootstock and the s...

AI Technical Summary

Problems solved by technology

At present, endogenous mRNA molecules that can be transmitted over long distances have been identified in fruit trees, including apple endogenous GAI (Xu et al., 2010), pear endogenous KN1 (Zhang Wenna, 2012), GAI (Zhang et al., 2012), NACP (Zhang et al., 2013), for the method of identifying whether the mRNA of herbaceous plant genes can be transmitted over a long distance, at present, only by constructing a vector carrying a specific tag for detection after transgene grafting (Haywood et al., 2005 ), microinjection detection of specific molecular probes on mRNA markers (Xoconostle-Cazares et al., 1999) and nested RT-PCR (Kanehira et al., 2010) and other techniques, although these techniques detect gene transmissibility compared Accurate, but the shortcomings are cumbersome process, high cost, high requirements, time-consuming, heavy workload, long working cycle, low efficiency, etc., which are difficult for general laboratories to complete

For perennial woody plants, RT-PCR-CAPS (Xu et al., 2010) is a relatively convenient method for studying the transfer of mRNA between rootstock and ear, but this method still has its shortcomings. The requirement for the gene sequence is high. For the mRNA that needs to be identified, there are natural differences in the gene sequence between the root and ear, and the general gene is difficult to meet the problem. Therefore, in the process of studying the transfer of mRNA between the root and ear, Its strict requirements greatly limit the number of mRNAs that can be identified, which makes it more difficult to carry out research on the grafting and transfer of endogenous mRNA in fruit trees, making it difficult to advance research on the long-distance transfer of mRNA in fruit trees. New long-distance transfer endogenous mRNA difficult to find

Images