阅读说明：本技术 黄瓜CsLsi2基因及其编码的蛋白和应用 (Cucumber CsLsi2 gene and its coded protein and application ) 是由 任华中 刘兴旺 张亚琦 董明明 翟许玲 尹帅 陈淑英 徐硕 冯钟萱 于 2019-10-14 设计创作，主要内容包括：本发明涉及黄瓜CsLsi2基因及其编码的蛋白和应用。所述黄瓜CsLsi2基因核苷酸序列如SEQ ID NO:1所示,该基因编码的蛋白序列如SEQ ID NO:2所示。本发明通过全基因组关联分析及筛选后克隆得到了与黄瓜果霜性状密切相关的黄瓜CsLsi2基因,该基因表达水平与黄瓜果霜含量呈正相关,而在黄瓜基因如SEQ ID NO:1所示序列的第754位的碱基由C变为T后会导致氨基酸翻译的提前终止。黄瓜CsLsi2基因的克隆及表达分析对解决黄瓜果霜基因资源难以筛选的问题起到了很大程度的促进作用。(The invention relates to a cucumber CsLsi2 gene and a protein coded by the same and application thereof. The nucleotide sequence of the cucumber CsLsi2 gene is shown as SEQ ID NO.1, and the protein sequence coded by the gene is shown as SEQ ID NO. 2. The cucumber CsLsi2 gene closely related to the cucumber frost character is obtained by cloning after whole genome correlation analysis and screening, the expression level of the gene is positively related to the cucumber frost content, and the early termination of amino acid translation can be caused after the 754 th base of the sequence shown in the cucumber gene SEQ ID NO.1 is changed from C to T. The cloning and expression analysis of the cucumber CsLsi2 gene play a great promoting role in solving the problem that cucumber frost gene resources are difficult to screen.)

1. The cucumber CsLsi2 gene is characterized in that the nucleotide sequence of the cucumber CsLsi2 gene is as follows:

i) 1, SEQ ID NO; or

ii) nucleotide sequences which are obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.1 and encode the same functional proteins.

2. The cucumber CsLsi2 gene encoded protein of claim 1, wherein the amino acid sequence of the protein is:

i) an amino acid sequence shown as SEQ ID No. 2; or

ii) the amino acid sequence shown in SEQ ID No.2 is substituted, deleted and/or added with one or more amino acids and has the same functional activity.

3. The primer pair for amplifying the cucumber CsLsi2 gene of claim 1, wherein the nucleotide sequence of the primer pair is as follows:

CsLsi2-F：5'-ATGGCCATGGATCATACTGTAA-3'

CsLsi2-R：5'-TCATTTTATAAGAACTAAACCA-3'。

4. the biological material containing the cucumber CsLsi2 gene of claim 1, wherein the biological material is an expression cassette, a recombinant expression vector, an engineering bacterium or a transgenic cell.

5. An expression inhibitor of a cucumber CsLsi2 gene is an agent capable of inhibiting expression of the cucumber CsLsi2 gene in a gene knockout or RNA interference mode.

6. Use according to claim 5, wherein the plant is a dicotyledonous plant, preferably a crop of the Cucurbitaceae family.

7. The cucumber CsLsi2 gene or the protein coded by the gene in the claim 1 is used for regulating the cucumber frost character.

8. The use of the cucumber CsLsi2 gene or the protein encoded by the gene in the reduction of the content of pericarp and frost in cucumber.

9. The application of claim 8, wherein the application is specifically to inhibit the expression of the cucumber CsLsi2 gene in cucumber, and preferably to mutate the nucleotide sequence 754 th site of the cucumber CsLsi2 gene from C to T as shown in SEQ ID NO. 1.

10. Use according to claim 8 or 9, wherein the cucumber is one or more of cucumber 3548-1, cucumber 3549-1, cucumber 3555-1, cucumber 3554-1, cucumber 3555-1 or cucumber 5553-1.

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a cucumber CsLsi2 gene and a protein coded by the same and application of the gene.

Background

Cucumber is an important source of dietary vitamin C. The method has the characteristics of high yield, good benefit and the like, is the existing main vegetable planting crop, and the quality of the cucumber directly determines the benefit of a producer. The appearance quality belongs to the quality category, and the fruit cream belongs to one of the important appearance qualities of the cucumber, and affects the sale of the cucumber to a great extent. Generally speaking, the variety with less fruit frost, bright and smooth melon strips is popular in the market. For a long time, there has been controversy about the composition and formation of cucumber fruit creams, and one idea is that: the formation of cucumber fruit cream is the result of the vertical skin-surface division of the single cell of the pericarp, and the hairy cell formed by the division of the single cell of the pericarp is further divided into two parts of a stalk and a granule; as the fruit grows, granulocytes gradually divide the sphere consisting of 6-8 cells filled with inclusions, and as the nucleus disappears, until the stage of commodity maturity, the inclusions secreted by the sphere are distributed together with the sphere on the cucumber fruit epidermis, the so-called fruit cream (pine, meizi, 1980). Another view is that: the cream is a silicide (Samules et al, 1993; Shenqiong, 2013). Although there is controversy that researchers have gradually become widely recognized through line production and physiological tests (such as grafting and spraying of exogenous silicon) to confirm that silicon influences cucumber frost formation, currently, gene mining and possible regulatory mechanisms related to frost formation are slow.

So far, the research on the influence of environmental factors and grafting on the formation of the fruit frost is concentrated, the genetic research on the fruit frost character is less, the research is mostly limited to the positioning of target genes and the development of molecular markers, and the results of different research groups are greatly different. The research of Shenqiong et al (2012) considers that the existence or the quantity of cucumber frost is controlled by 1 pair of major genes, and the existence or the quantity of the frost shows partial dominance; tiangui Li et al (2015) considered that the inheritance of cucumber frost characters conforms to 1 pair of additive major gene + additive-dominant polygene (D-2) models. The main reasons why this inconsistent findings have occurred may be: (1) the genetic background of the test materials adopted by each research group is different, and particularly, the cucumber seed material without fruit frost is almost not available; (2) the field survey standard of the fruit frost character is difficult to grasp, so that the difference of the research results among the fruit frost characters is large; (3) the influence of environmental conditions increases the difficulty of field investigation on the fruit frost characters, and the accuracy of data statistics is poor. The problems in the previous research fully explain the complexity of the research on the characters of the cucumber frost, so that the traditional means of genetic separation, map-based cloning, fine positioning and the like are adopted to excavate the genes related to the formation of the cucumber frost, which has a plurality of difficulties.

Disclosure of Invention

In order to solve the technical problems of difficult cloning and difficult improvement of cucumber genes in the prior art, the invention provides a cucumber CsLsi2 gene and a protein coded by the same and application of the cucumber CsLsi2 gene.

In a first aspect, the invention provides a cucumber CsLsi2 gene, wherein the nucleotide sequence of the cucumber CsLsi2 gene is as follows:

i) 1, SEQ ID NO; or

ii) nucleotide sequences which are obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.1 and encode the same functional proteins.

The invention further provides a protein coded by the cucumber CsLsi2 gene, wherein the amino acid sequence of the protein is as follows:

i) an amino acid sequence shown as SEQ ID No. 2; or

ii) the amino acid sequence shown in SEQ ID No.2 is substituted, deleted and/or added with one or more amino acids and has the same functional activity.

The invention further provides a primer pair for amplifying the cucumber CsLsi2 gene sequence, wherein the nucleotide sequence of the primer pair is as follows:

CsLsi2-F：5'-ATGGCCATGGATCATACTGTAA-3'

CsLsi2-R：5'-TCATTTTATAAGAACTAAACCA-3'。

the invention further provides an expression cassette containing the cucumber CsLsi2 gene.

The invention further provides a recombinant expression vector containing the cucumber CsLsi2 gene.

The invention further provides an engineering bacterium or a transgenic cell containing the cucumber CsLsi2 gene or the recombinant expression vector.

In a second aspect, the invention provides application of an expression inhibitor of a cucumber CsLsi2 gene in preparation of a plant with low content of frost, wherein the expression inhibitor of the cucumber CsLsi2 gene is a reagent capable of inhibiting expression of the cucumber CsLsi2 gene in a gene knockout or RNA interference mode.

Further, the plant is a dicotyledonous plant, preferably a crop of the cucurbitaceae family.

In a third aspect, the invention provides an application of the cucumber CsLsi2 gene or the protein coded by the gene in regulation of cucumber frost traits.

The invention further provides application of the cucumber CsLsi2 gene or the protein coded by the gene in reducing the content of pericarp and frost of cucumber.

Further, the application specifically comprises mutating the 754 th site of the nucleotide sequence shown as SEQ ID NO.1 in the cucumber gene from C to T.

Furthermore, the cucumber is one or more of cucumber 3548-1, cucumber 3549-1, cucumber 3555-1, cucumber 3554-1, cucumber 3555-1 or cucumber 5553-1.

The invention provides a cucumber CsLsi2 gene and a protein coded by the same and application thereof. The invention scans silicon related genes based on the physiological basis of character generation under the background of near-isogenic system materials and in the whole genome range, and clones the fruit frost related gene CsLsi2 by a method for detecting candidate genes and groups, wherein the gene is expressed in the advantages of pericarp and thorn, the expression level is related to the content of silicon, the expression level of CsLsi2 gene in cucumber strains with more fruit frost is high, and the expression level of CsLsi2 gene in cucumber strains with almost no fruit frost on the fruit pericarp is extremely low; the invention further clones the CsLsi2 gene of the cucumber in different fruit frost strains, and the base at the 754 th site of SEQ ID NO.1 is changed from C to T to cause the early termination of amino acid translation; the cloning and expression analysis of the cucumber CsLsi2 gene play a great promoting role in solving the problem that cucumber frost gene resources are difficult to screen.

Drawings

FIG. 1 is a comparison graph of differences of fruit skin frost of cucumber 3402 grafted by itself (3402/3402), cucumber 3402 grafted by black-seed pumpkin (3402/Cf) and cucumber 3402 grafted by northern nong's bright stock (3402/Cm), wherein B, B1, B2, C, C1 and C2 are results under scanning of an electron microscope, provided in example 1 of the present invention;

FIG. 2 is a schematic diagram showing the changes of the fruit skin frost of the cucumber 3402 grafted by itself (3402/3402), the cucumber 3402 grafted by black-seed pumpkin (3402/Cf) and the cucumber 3402 grafted by northern agricultural bright stock (3402/Cm) provided in example 1 of the present invention;

FIG. 3 is a schematic diagram of the change of the silicon content of the fruit after silicon application and silicon absorption inhibition of high-generation inbred lines 3548-1 and 3549-1 of cucumber according to example 2 of the present invention;

FIG. 4 is a comparison of cucumber 3548-1 and cucumber 3549-1 fruit creams provided in example 3 of the present invention;

FIG. 5 is a graph comparing the silicon content of the peel and flesh of cucumbers 3548-1 and 3549-1 provided in example 3 of the present invention;

FIG. 6 is a diagram showing the results of the energy spectrum detection and analysis of the pericarp of cucumber 3548-1 and cucumber 3549-1 provided in example 3 of the present invention;

FIG. 7 is a schematic diagram showing changes in expression levels of cucumber CsLsi1/CsiT-1 gene and CsiT-2 gene in cucumber 3549-1, cucumber 3548-1, cucumber 3555-1, cucumber 3554-1, cucumber 5553-1 and cucumber 5552-1, which are provided in example 4 of the present invention;

FIG. 8 is a comparative clone map of the CsLsi2 gene of Cucumis sativus provided in example 4 of the present invention in Cucumis sativus 3549-1, Cucumis sativus 3548-1, Cucumis sativus 3555-1, Cucumis sativus 3554-1, Cucumis sativus 5553-1 and Cucumis sativus 5552-1;

FIG. 9 is a diagram showing the analysis of the protein transmembrane region of the CsLsi2 gene of Cucumis sativus provided in example 5 of the present invention;

FIG. 10 is a graph showing the analysis of the expression of the CsLsi2 gene of Cucumis sativus in roots, stems, leaves, pistils, stamens, fruits, pericarps and pulps in Cucumis sativus 3548-1 and Cucumis sativus 3549-1, according to example 6 of the present invention;

FIG. 11 is a diagram showing an analysis of the expression of the CsLsi2 gene of Cucumis sativus provided in example 6 of the present invention in the pericarp of Cucumis sativus 3549-1, Cucumis sativus 3548-1, Cucumis sativus 3555-1, Cucumis sativus 3554-1, Cucumis sativus 5553-1 and Cucumis sativus 5552-1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental methods used in the examples of the present invention are conventional experimental means unless otherwise specified, and the cucumber high-generation inbred lines 3548-1, 3549-1, 3555-1, 3554-1, 5553-1 and 5552-1 (hereinafter, abbreviated as cucumber 3548-1, cucumber 3549-1, cucumber 3555-1, cucumber 3554-1, cucumber 5553-1 and cucumber 5552-1) used in the examples are from the group of cucumber genetic improvement subjects of the university of agriculture in China, which are disclosed in Plant Biotechnology Journal (Yang et al, 2019,17:289 301), and can be publicly obtained (only for scientific research and teaching purposes).

Wherein the cucumber 3548-1 is a mutant of cucumber 3549-1 (less cucumber 3548-1 frost), the cucumber 3554-1 is a mutant of cucumber 3555-1 (less cucumber 3554-1 frost), and the cucumber 5552-1 is a mutant of cucumber 5553-1 (less cucumber 5552-1 frost).