CN-122012555-A - Isolated nucleic acid molecule, recombinant bacterium and application thereof in cultivation of high-sugar tobacco variety

Abstract

The invention discloses an isolated nucleic acid molecule, recombinant bacteria and application thereof in cultivation of high-sugar tobacco varieties, and relates to the technical field of genetic improvement of tobacco quality. According to the invention, a NtTD gene specifically expressed in the glandular Mao Xian is cloned from tobacco, after the NtTD gene of a flue-cured tobacco variety K326 is knocked out by a CRISPR/Cas9 gene editing technology, the content of total sugar and reducing sugar in tobacco leaves is obviously increased after a homozygous mutant tobacco material of the NtTD1 gene is baked, and the gene knocked out has no influence on the development of tobacco plants, so that the method has important utilization value in cultivating high-sugar tobacco varieties, and is beneficial to improving the quality of tobacco leaves.

Inventors

• CHANG AIXIA
• YANG XINGYOU
• MA PENG
• LONG TAN
• YAN MIN
• WANG DONG
• Zhang Xuanhuai
• MAO MIN
• YANG XUE
• LUO WANLIN
• ZHANG YU
• QIAN YU
• WANG YANQI
• HU QINGZHU
• LI YITING
• JIANG HONG
• FENG WENLONG

Assignees

• 中国烟草总公司四川省公司
• 四川省烟草公司凉山州公司
• 四川省烟草公司宜宾市公司
• 四川省烟草公司广元市公司
• 四川省烟草公司攀枝花市公司

Dates

Publication Date

20260512

Application Date

20260225

Claims (10)

1. 1. An isolated nucleic acid molecule having the nucleotide sequence set forth in SEQ ID No. 1.
2. 2. A vector comprising the isolated nucleic acid molecule of claim 1.
3. 3. A gRNA molecule that targets the isolated nucleic acid molecule of claim 1.
4. 4. The gRNA molecule according to claim 3, comprising a nucleotide sequence as set forth in SEQ ID NO. 2 or SEQ ID NO. 3.
5. 5. A knockout vector comprising a gRNA molecule of any one of claims 3-4.
6. 6. A recombinant bacterium comprising the gene knockout vector of claim 5; Preferably, the recombinant bacterium is escherichia coli or agrobacterium.
7. 7. Use of the isolated nucleic acid molecule of claim 1, the vector of claim 2, the gRNA molecule of any one of claims 3-4, the gene knockout vector of claim 5, or the recombinant bacterium of claim 6 for breeding high sugar tobacco varieties.
8. 8. The application of claim 7, wherein the application comprises at least one of the following application modes: (1) Setting a gene knockout target point for the isolated nucleic acid molecule of claim 1, constructing a gene knockout vector, and then transferring the gene knockout vector into target tobacco; (2) Constructing a gene knockout vector comprising the gRNA molecule of any one of claims 3-4, and then transferring the gene knockout vector into tobacco of interest; (3) Transferring the gene knockout vector of claim 5 into a tobacco of interest; (4) When the recombinant bacterium of claim 6 is agrobacterium, infecting the agrobacterium with the tobacco of interest; Preferably, a gene knockout target is set for at least one of exons 1 to 8 of the isolated nucleic acid molecule of claim 1, a gene knockout vector is constructed, and then the gene knockout vector is transferred into tobacco of interest; preferably, a gene knockout target is set for exon 1 of the isolated nucleic acid molecule of claim 1, a gene knockout vector is constructed, and then the gene knockout vector is transferred into tobacco of interest.
9. 9. A method for increasing the sugar content of tobacco, characterized in that it comprises any one of the following modes i-v: (i) Knocking out NtTD gene in tobacco; (ii) Setting a gene knockout target point for the isolated nucleic acid molecule of claim 1, constructing a gene knockout vector, and then transferring the gene knockout vector into target tobacco; (iii) Constructing a gene knockout vector comprising the gRNA molecule of any one of claims 3-4, and then transferring the gene knockout vector into tobacco of interest; (iv) Transferring the gene knockout vector of claim 5 into a tobacco of interest; (v) When the recombinant bacterium of claim 6 is agrobacterium, the agrobacterium is infected with the tobacco of interest.
10. 10. The method of claim 9, wherein the tobacco sugar content is tobacco reducing sugar and/or tobacco total sugar content; preferably, the target tobacco is selected from flue-cured tobacco variety K326, RG17, TN86, basma, zhongyan 100, yuan tobacco No. 10, hunan tobacco No. 7, yunyan tobacco 85, yunyan tobacco 87, NC89 or Honghuadajinyuan.

Description

Isolated nucleic acid molecule, recombinant bacterium and application thereof in cultivation of high-sugar tobacco variety Technical Field The invention relates to the technical field of genetic improvement of tobacco quality, in particular to an isolated nucleic acid molecule, recombinant bacteria and application thereof in cultivation of high-sugar tobacco varieties. Background Tobacco is an important cash crop in China, and the chemical components of the cured tobacco are main factors for determining the intrinsic quality and the use value of the cured tobacco. Among the chemical components, the content of total sugar and reducing sugar in the cured tobacco leaf, the ratio and the difference value thereof are one of key chemical indexes for measuring the internal quality and industrial availability of the tobacco leaf, and have a vital effect on improving the sensory and style characteristics of the cigarette product. The contribution of sugar substances to tobacco quality is mainly represented by (1) improving the taste and aroma quality of tobacco leaves. The sugar substances in the tobacco leaves can generate acid reaction during combustion and suction, so that alkaline substances in the smoke can be neutralized, the irritation is reduced, and the smoke is more mellow and smooth. The reducing sugar and the total sugar content are obviously positively correlated with the aroma quantity, aroma quality, concentration and other quality indexes of the tobacco leaves. When the ratio of the two sugars (reducing sugar/total sugar) is increased and the difference of the two sugars is reduced, the aroma and the concentration of the tobacco leaves are obviously improved. (2) improving the appearance quality. Tobacco leaves with a high sugar content generally have a better quality of appearance. Researches show that the disaccharide ratio is extremely obviously and positively correlated with the appearance quality indexes such as the color shade, the maturity, the softness, the oily feel and the like of the tobacco leaves. The sugar substance can also make the tobacco leaf softer, rich in elasticity, bright in color and luster, and resistant to pressure and difficult to break. (3) promote the formation of aroma substances. The saccharides are important precursors for forming aroma substances, and under the high temperature condition of above 300 ℃, the saccharides can be pyrolyzed singly to form carbonyl compounds such as furan derivatives, ketones, aldehydes and the like. In addition, the saccharide and the amino acid undergo Maillard reaction, so that various aroma substances can be generated, and unique aroma characteristics of tobacco can be endowed. Therefore, the method for improving the reducing sugar and the total sugar content of the cured tobacco leaves is explored, and has important significance for improving the quality of the tobacco leaves. Methods for increasing the sugar content of tobacco leaves generally comprise three modes of cultivation, baking and breeding. In the aspect of cultivation and baking, the cultivation and the baking are generally performed in modes of optimizing a planting and harvesting method, accurately fertilizing, adjusting carbon and nitrogen in soil, optimizing baking technology and the like, but the modes have higher requirements on technical standards, are higher in operation difficulty, are not mastered in place or are not done in place by tobacco farmers, and are difficult to achieve effective results. The cultivation of the variety with high sugar content by the genetic breeding mode is the most economical and stable mode, however, as sugar metabolism is primary metabolism, the direct regulation of genes synthesized by sugar metabolism can possibly cause abnormal development of tobacco, so that the research of improving the sugar content by regulating the specific target genes is still fresh. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide an isolated nucleic acid molecule, recombinant bacteria and application thereof in cultivating high-sugar tobacco varieties so as to improve the sugar content of the tobacco varieties and improve the tobacco quality. The invention is realized in the following way: In a first aspect, the invention provides an isolated nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO. 1. In a second aspect, the invention provides a vector comprising an isolated nucleic acid molecule. In a third aspect, the invention provides a gRNA molecule that targets an isolated nucleic acid molecule. In a fourth aspect, the invention provides a gene knockout vector comprising a gRNA molecule as described above. In a fifth aspect, the present invention provides a recombinant bacterium comprising the above gene knockout vector. In a sixth aspect, the invention provides the use of an isolated nucleic acid molecule, vector, gRNA molecule, gene knockout vector or recombinant bacteriu