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CN-122012576-A - Gene MdLAC for enhancing saline-alkali resistance of apples, extraction method and system

CN122012576ACN 122012576 ACN122012576 ACN 122012576ACN-122012576-A

Abstract

The invention belongs to the technical field of molecular biology and discloses a gene MdLAC for enhancing the salt and alkali resistance of apples and application thereof, wherein the extraction method of the gene MdLAC for enhancing the salt and alkali resistance of apples comprises the following steps of firstly extracting total RNA from root systems of M9-T337 of apple stocks, taking cDNA obtained by reverse transcription as a cloning template, secondly designing MdLAC12 primers according to GDDH13 of an apple genome database, carrying out conventional polymerase chain reaction PCR, thirdly, carrying out homologous recombination on a PCR product obtained MdLAC12 and pMD18-T, converting a connection product into competent cells of DH5 alpha of escherichia coli, and carrying out sequencing analysis on resistance bacterial plaques to obtain CDs sequences of MdLAC 12. The invention is helpful for breeding excellent saline-alkali resistant apple stocks, and can also be used for improving the saline-alkali soil utilization in the eugenic zone. The method has the advantages of simple operation, strong saline-alkali resistance and low production cost, and can be widely used in the process of breeding saline-alkali resistant germplasm resources.

Inventors

  • ZUO XIYA
  • TIAN XUAN
  • LI SHAOHUAN
  • ZHANG DONG
  • SHAO YUN
  • ZHANG XIAOYUN
  • MA JUANJUAN
  • JING MINGYUAN
  • KANG ZHIWEI
  • Zeng Fanman

Assignees

  • 西北农林科技大学

Dates

Publication Date
20260512
Application Date
20260109

Claims (7)

  1. 1. The application of MdLAC gene to enhancing salt and alkali resistance of apples is characterized in that the nucleotide sequence of MdLAC gene is shown as SEQ ID NO. 1.
  2. 2. The use of MdLAC genes to enhance salt and alkaline tolerance of apples according to claim 1, wherein the interfering sequence of MdLAC genes is shown in SEQ ID No. 2.
  3. 3. A method for extracting MdLAC genes for enhancing the salt and alkali resistance of apples, which is characterized by comprising the following steps: Step one, extracting total RNA from the root system of an apple stock M9-T337, and taking cDNA obtained by reverse transcription as a cloning template; designing MdLAC12 primers according to the GDDH13 of the apple genome database, and performing conventional Polymerase Chain Reaction (PCR); And thirdly, carrying out homologous recombination on the PCR product of MdLAC and pMD18-T, converting the connection product into competent cells of escherichia coli DH5 alpha, picking up resistant bacterial plaques, and carrying out sequencing analysis to obtain the CDs sequence of MdLAC.
  4. 4. The method for extracting MdLAC gene for raising salt and alkali tolerance of apple according to claim 3, wherein the obtained CDs sequence of MdLAC12 is introduced into pCambia2300 vector by means of homologous recombination, the constructed vector is transferred into agrobacterium for genetic transformation of apple, and one segment of sequence in clone MdLAC sequence is inserted into pK7GWIWG2D interference vector, and transferred into agrobacterium for genetic transformation of apple.
  5. 5. The method for extracting MdLAC gene for enhancing salt and alkali resistance of apples according to claim 3, wherein GL-3 tissue culture seedling leaves of 30 d after subculture are selected as transgenic materials, a transgenic apple strain is obtained by using an agrobacterium-mediated transformation system, DNA and RNA level detection is carried out on apple resistant buds, and positive transgenic apple strains are screened.
  6. 6. The method for extracting MdLAC gene for enhancing the salt and alkali resistance of apples according to claim 3, wherein 3 MdLAC12 over-expression lines, 2 MdLAC interference lines and GL-3 are subjected to subculture, plants grown to 2.0 cm are selected for rooting culture after the subculture is carried out to a certain number, 30 d plants are cultured in rooting culture medium and then transferred into a nutrition pot, 60 d plants are grown, and relevant physiological indexes are counted after 60 d plants are treated.
  7. 7. A system for performing the method of extracting MdLAC gene enhancing saline-alkali tolerance of apples according to any one of claims 3 to 6, said system comprising: The reverse transcription module is used for extracting total RNA from the root system of the apple stock M9-T337, and taking cDNA obtained by reverse transcription as a cloning template; the chain reaction module is used for designing MdLAC primers according to the apple genome database GDDH13 and carrying out conventional polymerase chain reaction PCR; And the sequencing analysis module is used for carrying out homologous recombination on the PCR product of MdLAC and pMD18-T, converting the connection product into E.coli DH5 alpha competent cells, and carrying out sequencing analysis on the resistance bacterial plaque to obtain the CDs sequence of MdLAC.

Description

Gene MdLAC for enhancing saline-alkali resistance of apples, extraction method and system Technical Field The invention belongs to the technical field of molecular biology, and particularly relates to a gene MdLAC for enhancing the salt and alkali resistance of apples, an extraction method and a system. Background Apple (Malus domestca borkh.) is planted in temperate regions of five continents, and is one of the most important tree species of deciduous fruit trees in the world. According to the national statistical bureau data, the total apple cultivation area in China reaches 208.8 ten thousand hectares, the total yield is 44.6.6 ten thousand tons (the national statistical bureau data 2021 of the people's republic of China), the ranking world is first, and the method plays an important role in the development of fruit industry in China. The total amount of cultivated land in China is less, the quality is not high, the reserve resources are insufficient, and the relationship of developing grain production and exerting comparative benefits must be treated. Under the premise of ensuring the basic self-sufficiency of grains and absolute safety of ration, the apple planting industry is better developed, new apple planting land needs to be developed, the northwest loess plateau is used as an apple eugenic zone, black soil with more salt is used as a main material, the salt in the soil is increased due to long-term unreasonable irrigation and single fertilizer application in production, if the water evaporates in the hollow zone, the salt is left, and a saline-alkali zone (Zhang Rui 2021) is formed due to long-term accumulation. At present, more than 70% of the soil in loess plateau areas is saline-alkali soil (mainly containing NaCl and NaHCO3 salts and having pH of 8.0-8.5) (Xue Hao and the like 2015), most of the soil is unsuitable for grain use, the apple has strong adaptability to the soil, and the slightly saline-alkali soil can be cultivated in a piece after being properly improved, but the severe saline-alkali soil is high in cost, long in time consumption and poor in effect and is generally unsuitable for use (Shan Shuangquan 2017). In production, the method has the advantages that in the symbiont formed by the scion and the stock, the stock with strong resistance directly influences the adaptability of the scion to adverse conditions (Moore 1984), so that the stock with strong salt-alkali resistance is the most direct and effective method for coping with soil salinization in production, and the method for screening the saline-alkali resistant stock variety is significant. Through the above analysis, the problems and defects existing in the prior art are as follows: over 70% of the soil in loess plateau areas is saline-alkali soil (mainly containing NaCl and NaHCO3 salt and having pH of 8.0-8.5) (Xue Hao and the like 2015), most of the soil is unsuitable for grain, the apple has strong adaptability to the soil, and the slightly saline-alkali soil can be cultivated in a piece after being properly improved, but the severe saline-alkali soil is high in cost, long in time consumption and poor in effect and is generally not suitable for improvement. Disclosure of Invention Aiming at the problems existing in the prior art, the invention provides a gene MdLAC for enhancing the salt and alkali resistance of apples and application thereof, wherein the nucleotide sequence of MdLAC is SEQ ID NO 1 and MdLAC is an interference sequence table SEQ ID NO 2. The invention is realized in such a way that the extraction method of the gene MdLAC for enhancing the salt and alkali resistance of apples comprises the following steps: Step one, extracting total RNA from the root system of an apple stock M9-T337, and taking cDNA obtained by reverse transcription as a cloning template; designing MdLAC12 primers according to the GDDH13 of the apple genome database, and performing conventional Polymerase Chain Reaction (PCR); And thirdly, carrying out homologous recombination on the PCR product of MdLAC and pMD18-T, converting the connection product into competent cells of escherichia coli DH5 alpha, picking up resistant bacterial plaques, and carrying out sequencing analysis to obtain the CDs sequence of MdLAC. Further, the CDs sequence MdLAC is introduced into pCambia2300 vector through homologous recombination, the constructed vector is transferred into agrobacterium for apple genetic transformation, and one segment of sequence in the clone MdLAC sequence is inserted into pK7GWIWG2D interference vector and transferred into agrobacterium for apple genetic transformation. Further, selecting GL-3 tissue culture seedling leaves of 30 d after subculture as a transgenic material, obtaining a transgenic apple strain by using an agrobacterium-mediated transformation system, detecting the obtained apple resistant buds at DNA and RNA levels, and screening out a positive transgenic apple strain. Further, the obtained 3 MdLAC over-expression