CN112760334B - Gene for regulating and controlling sugar content of tomato fruit and application thereof - Google Patents

Gene for regulating and controlling sugar content of tomato fruit and application thereof Download PDF

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CN112760334B
CN112760334B CN202110063588.5A CN202110063588A CN112760334B CN 112760334 B CN112760334 B CN 112760334B CN 202110063588 A CN202110063588 A CN 202110063588A CN 112760334 B CN112760334 B CN 112760334B
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tomato
slcin2
gene
sugar content
regulating
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CN112760334A (en
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姜晶
张琼琼
刘欣
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Shenyang Agricultural University
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    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

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Abstract

The invention relates to the fields of biotechnology and genetic breeding, in particular to a gene for regulating and controlling the sugar content of tomato fruits and application thereof. The gene for regulating and controlling the sugar content of the tomato fruits is SlCIN2, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1. Firstly, extracting tomato RNA, carrying out reverse transcription on cDNA, designing an amplification primer, and amplifying an SlCIN2 gene by taking the cDNA as a template; the gene SlCIN2 is constructed into a pB7GWIWG2 vector by using gateway technology, correct positive clones are screened, the obtained positive clones are transformed into an LBA4404 agrobacterium-infected state to obtain recombinant agrobacterium tumefaciens, and the recombinant agrobacterium tumefaciens is transformed into a tomato to obtain a tomato plant with improved fruit sugar content.

Description

Gene for regulating and controlling sugar content of tomato fruit and application thereof
Technical Field
The invention relates to the fields of biotechnology and genetic breeding, in particular to a gene for regulating and controlling the sugar content of tomato fruits and application thereof.
Background
The fruit quality of tomatoes consists of appearance quality, flavor quality, nutritional quality and processing and storage quality. Flavor attributes include, among others, sweetness, sourness, aroma, etc., associated with soluble sugars, organic acids and volatile aromatic compounds in the fruit. Loss of flavour can seriously affect the organoleptic quality of the fruit and thus its economic value. The improvement of the sweetness of tomato fruits is an important link for improving the quality of tomatoes, the sucrose content in the fruits of most tomato varieties is very low, mainly glucose and fructose, and the glucose and fructose content in the tomato fruits is in positive correlation with the activity of invertase. In the tomato fruit development process, sucrose metabolism is particularly important for the growth and development and quality formation of tomato fruits.
Cytoplasmic Invertases (CINs) are a family of proteins present in plants that obligately break down sucrose, playing an important role in regulating plant organ development and product quality. The cytoplasmic invertase At-A/N-InvE in Arabidopsis regulates seedling growth by controlling the ratio of sucrose to hexose content. The excessive expression of the cytoplasm invertase gene in the taxus chinensis suspension cells can promote the sugar metabolism level, thereby causing the synthesis of secondary metabolite taxane. In the research on populus trichocarpa, the activity of a cytoplasmic invertase CIN is found to play an important role in cellulose biosynthesis of wood. The discovery of alkaline/neutral invertase activity in peach trees may play an important role in controlling fruit sugar content and fruit growth and development. Cytoplasmic invertase activity is higher in immature fruits of radish and sugarcane, and as the fruit maturity activity is reduced to a minimum, the reduction of invertase activity favors the accumulation of sucrose. However, few studies have reported the mechanism of action of cytoplasmic convertase on the sugar metabolism of tomato fruits.
There are 8 members of the cytoplasmic convertase family in tomato, of which SlCIN2 is mainly expressed during the soluble sugar accumulation stage late in fruit development. The expression level of SlCIN2 in tomatoes is reduced by an RNA interference technology, and the soluble sugar content of tomato fruits is improved on the premise of not influencing the tomato plant types.
Disclosure of Invention
One of the purposes of the invention is to provide a gene for regulating and controlling the sugar content of tomato fruits, wherein the gene is SlCIN2, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1.
The other purpose of the invention is to provide a protein for regulating and controlling the sugar content of tomato fruits, wherein the protein is coded by SlCIN2 gene.
The invention also aims to provide an RNAi vector of a target SlCIN2 gene, which is prepared by the following steps:
extracting tomato RNA, carrying out reverse transcription on cDNA, designing an amplification primer, and amplifying an SlCIN2 gene by taking the cDNA as a template; the gene SlCIN2 is constructed into a pB7GWIWG2 vector by using gateway technology to obtain a recombinant plasmid pB7GWIWG-SlCIN2, namely an RNAi vector of a target gene SlCIN 2.
Further, the nucleotide sequence of the amplification primer is as follows:
SlCIN2-F:5'-CGCACCTGGTCCATTGTGTCGTCTT-3'；
SlCIN2-R:5'-GGTGTGTTCTTAGGGTCGCT-3'。
the fourth object of the present invention is to provide a recombinant Agrobacterium comprising the RNAi vector.
The fifth purpose of the invention is to provide application of the SlCIN2 gene in improving sugar content of tomato fruits.
The invention aims at providing a method for improving the sugar content of tomato fruits, which is characterized by comprising the following steps:
transferring the recombinant agrobacterium into tomato to obtain tomato plant with increased fruit sugar content.
Further, the tomato line is Micro-Tom.
Compared with the prior art, the invention has the following beneficial effects:
the recombinant plasmid pB7GWIWG-SlCIN2 is transferred into tomato plants by an agrobacterium-mediated method to inhibit expression of SlCIN2, so that the technology changes the sugar content of tomato fruits, has important influence on the fruit quality of fruit vegetables, and can be used for cultivating plant varieties with high fruit sugar content.
Drawings
FIG. 1 shows the electrophoresis detection of pB7GWIWG2-SlCIN2 vector construction process, wherein A: amplifying silent target fragments by PCR; m: DNA Marker 2000; h2O: negative control; 1-5: PCR amplification products; b: carrying out PCR detection on TOPO reaction bacterial liquid; m: DNA Marker 1000; 1-4: PCR amplification products; c: PCR detection of LR reaction bacterial liquid; m: DNA Marker 1000; 1-6: PCR amplification products; d: carrying out PCR detection on the agrobacterium tumefaciens transformed liquid; m: DNA Marker 1000; 1-5: and (5) PCR amplification products.
FIG. 2 is the acquisition of transgenic tomato plants, in which A: aseptic seedlings; b: co-culturing; c: culturing the sprouts; d: rooting culture; e: hardening seedlings; f: and (5) transplanting.
Fig. 3 is electrophoretic detection of SlCIN3 transgenic plants, wherein M: DNA Marker 2000; h2O: negative control; CK: a wild-type plant; 1-10: and (3) transgenic plants.
FIG. 4 is an expression analysis of leaf blades of SlCIN2 silenced plants, wherein CK: a wild-type plant; 1-7: silencing a transgenic plant; indicates a significant level of difference (P <0.05) and indicates a very significant level of difference (P < 0.01).
Fig. 5 is an expression analysis of the SlCIN2 in the process of fruit ripening of silenced plants, wherein the difference indicates a significant level (P <0.05) and the difference indicates a very significant level (P < 0.01).
Fig. 6 shows the soluble solids content of the red-ripe fruit of the plants silenced by SlCIN2, wherein the difference is significant (P <0.05) and the difference is extremely significant (P < 0.01).
Fig. 7 shows the soluble sugar content of the red-ripe fruit of the plants silenced by SlCIN2, wherein the difference is shown to be significant (P <0.05) and the difference is shown to be extremely significant (P < 0.01).
Detailed Description
The invention is described in detail below with reference to the drawings and specific examples, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
RNAi vector and construction of recombinant Agrobacterium
And (3) constructing an SlCIN2-RNAi vector by using gateway technology.
1. Extracting tomato RNA, carrying out reverse transcription to obtain cDNA, amplifying a target fragment SlCIN2 gene by using the cDNA as a template, wherein the size of the target fragment is 170bp (shown in figure 1A), and the nucleotide sequence of the target fragment is shown in SEQ ID No. 1;
the primer sequences are as follows:
SlCIN2-F:5'-CGCACCTGGTCCATTGTGTCGTCTT-3', SEQ ID NO. 2.
SlCIN2-R is shown in 5'-GGTGTGTTCTTAGGGTCGCT-3', SEQ ID NO. 3.
The amplification reaction system is as follows:
10 mu L of high fidelity DNA polymerase, 1 mu L of SlCIN2-F, 1 mu L of SlCIN2-R, 2 mu L of cDNA and 6 mu L of RNase-Free ddH2O 6.
The amplification reaction procedure was as follows:
30s at 98 ℃, 1 cycle; 35 cycles of 98 ℃ for 10s,55 ℃ for 20s, and 72 ℃ for 10 s; 7min at 72 ℃.
2. Through TOPO reaction, 4. mu.L of the target fragment amplification product, 1. mu.L of pENTR/D-TOPO intermediate vector plasmid, 1. mu.L of salt solution were ligated in a metal bath at 25 ℃ for 8 h. The target fragment was ligated with the intermediate vector TOPO vector and the intermediate clone was obtained by E.coli transformation (FIG. 1B). Then transferring the target fragment into a final vector pB7GWIWG2 vector by using LR reaction, pENTR/D-TOPO-SlCIN 36 mu L, the final vector pB7GWIWG2(I), plasmid 2 mu L and LR close II 2 mu L react at 25 ℃ for 8h, and then adding salt solution to stop the reaction. Transformed into escherichia coli DH5 alpha competence, single colony is picked for bacteria liquid PCR identification (figure 1C), and correct positive clone is obtained by sequencing comparison. The obtained positive clones were transformed into LBA4404 Agrobacterium competence to obtain recombinant Agrobacterium (FIG. 1D) for subsequent tomato genetic transformation.
Example 2
Recombinant agrobacterium transformed tomato
And (3) transforming the recombinant vector plasmid into the tomato by a leaf disc method by using the Micro-Tom tomato as a test material to obtain a stable genetic transformation material.
When tomato seedlings in the culture medium grow until cotyledons are completely expanded (figure 2A), cutting 0.5cm cotyledons, placing in a pre-culture medium, culturing in dark for 2d, and culturing with OD600The agrobacterium tumefaciens reaching 0.6-0.8 infects the tomato cotyledon, the back of the infected leaf is upward and placed in a co-culture medium (figure 2B), and after dark culture for 2d, the explant is transferred to an antibacterial culture medium for culture. When the explants were grown in this medium for two weeks, they were transferred to germination medium for culture (FIG. 2C), after which the medium was changed two weeks apart. When the adventitious bud grows to 2-3cm, the adventitious bud is cut from the stem base and cultured in a rooting medium (FIG. 2D). After adventitious buds grow roots, taking out the complete plant from a culture bottle, putting the plant into Kawasaki nutrient solution for hardening seedlings (figure 2E), transplanting the plant into a nutrition pot for culturing after the plant adapts to the external environment, and obtaining a silent SlCIN2 transgenic tomato plant (figure 2F).
Example 3
Identification of silent SlCIN2 transgenic tomato plants
And extracting DNA of the transgenic tomato leaves, and performing PCR identification on the screened silent SlCIN2 transgenic plants. SlCIN2 silent plants were tested by using Bar gene locus design specific primers on pB7GWIWG2 vector plasmid.
The specific primer sequences are as follows:
Bar-F: 5'-GAAGTCCAGCTGCCAGAAA-3', as shown in SEQ ID NO. 4.
Bar-R: 5'-CACCATCGTCAACCACTACA-3' T, as shown in SEQ ID NO. 5.
And carrying out PCR amplification by taking DNA as a template and carrying out electrophoresis detection, wherein the detected specific fragment length is 439 bp. As a result, it was found that a 439bp specific fragment was detected in all 10 transgenic lines (FIG. 3), while the negative control (ddH2O and non-transformed wild type Micro-Tom) did not amplify any band. The 10 strains are all transgenic positive plants and can be used for subsequent experimental analysis.
Example 4
Analysis of expression quantity of SlCIN2 in silent SlCIN2 transgenic tomato plant leaves in order to definitely silence the expression level of SlCIN2 in a SlCIN2 transgenic line, tender leaves of identified positive plants are taken, normal Micro-Tom tomatoes are taken as a control, and RNA is extracted and is reversely transcribed into cDNA. qRT-PCR analysis was performed using real-time quantitative primers from SlCIN2, with the Actin gene as the internal reference.
The real-time quantitative primer sequences are as follows:
qslicin 2F: 5'-AGCGACCCTAAGAACACACC-3', as shown in SEQ ID NO. 6.
qslicin 2R: 5'-CCCAGAATAGCAAGGAAGC-3', as shown in SEQ ID NO. 7.
As shown in FIG. 4, the expression level in the leaves of the silent transgenic lines is remarkably reduced by 1.8-9.1 times compared with the control, which indicates that the SlCIN2 silent vector plasmid is successfully transformed into tomato and can inhibit the expression of SlCIN2 in the leaves. The No.3, 4 and 7 strains show extremely obvious difference, and compared with a control, the expression quantity of the No.3 strain SlCIN2 is reduced by about 4 times, the expression quantity of the No.4 strain is reduced by 5 times, and the expression quantity of the No.7 strain is reduced by 9.1 times; therefore, the three strains with higher silencing efficiency are selected for subsequent experiments.
Example 5
Analysis of expression level of SlCIN2 in fruits of silent SlCIN2 transgenic tomato plants
According to the analysis result of the leaf expression of the silent strain, selecting fruits of No.3, 4 and 7 strains in green maturity, color transition and red maturity, extracting RNA and performing reverse transcription to obtain cDNA by taking normal Micro-Tom tomatoes as a control, and analyzing the change of the expression of the silent transgenic tomato fruits in different development stages by using a real-time quantitative primer of SlCIN2 and an Actin gene as an internal reference and performing qRT-PCR. As can be seen from FIG. 5, the expression levels of the three different fruit development stages are similar in the change trend, the green-ripe stage expression is low, and the expression levels are increased in the color-conversion stage and the red-ripe stage, regardless of the control CK or the transgenic line; however, compared with CK, the expression level of the fruits of the silent SlCIN2 transgenic line is reduced in different development stages. The expression quantity change of the three transgenic strain lines in the green-maturing stage is relatively small, and only the No.4 strain line is obviously reduced compared with CK in the green-maturing stage; expression analysis in the color transition period of tomato fruits shows that compared with a control CK, the expression levels of the three strains are remarkably reduced by 1.8 times, 2.9 times and 3.4 times respectively; the expression in the red mature period of the fruit is similar to that in the color conversion period, and the transgenic lines are greatly reduced compared with a control, wherein the reduction multiple of the No.3 line is the highest. The results show that the expression level of SlCIN2 is obviously reduced in fruits of silent SlCIN2 tomato plants in different developmental stages.
Example 6
Determination of sugar content in fruits of silent SlCIN2 transgenic tomato plants
And performing primary phenotype identification on the early-stage screened transgenic positive strains, and selecting transgenic homozygous T2-generation strains subjected to PCR identification for index determination. To clarify the mechanism of action of SlCIN2 in the sugar metabolism process of tomato fruits, we measured the soluble solids content of the fruits in the red-ripe stage of transgenic tomatoes. As can be seen from fig. 6, the soluble solids content in the red-ripe fruit of transgenic tomato was significantly increased. Further, the content of soluble sugar in the tomato red-ripe fruit is determined by using a high performance liquid chromatography analysis method, and the result is shown in fig. 7, and the contents of fructose, glucose and sucrose in the fruits of the silent SlCIN2 transgenic lines are all obviously increased.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
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Claims (1)

1.SlCIN2Application of gene in increasing sucrose content in tomato fruit, characterized in thatSlCIN2The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the tomato is silentSlCIN2Transgenic tomato plants are homozygous for the T2 generation.
CN202110063588.5A 2021-01-18 2021-01-18 Gene for regulating and controlling sugar content of tomato fruit and application thereof Expired - Fee Related CN112760334B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103789346A (en) * 2014-02-14 2014-05-14 浙江农林大学 Loquat vacuole invertase, and method for improving tobacco sugar level employing encoding gene thereof
CN111748556A (en) * 2020-07-24 2020-10-09 沈阳农业大学 Application of SlmiR319b in regulation and control of tomato plant type, recombinant plasmid, recombinant strain and transgenic strain

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1183379A2 (en) * 1999-06-12 2002-03-06 Thomas Dr. Roitsch Promoter system and production and use of the same
CN104774869A (en) * 2015-04-20 2015-07-15 重庆大学 Method for cultivating long-storage tomato plants by virtue of silent tomato gene SlNZG

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103789346A (en) * 2014-02-14 2014-05-14 浙江农林大学 Loquat vacuole invertase, and method for improving tobacco sugar level employing encoding gene thereof
CN111748556A (en) * 2020-07-24 2020-10-09 沈阳农业大学 Application of SlmiR319b in regulation and control of tomato plant type, recombinant plasmid, recombinant strain and transgenic strain

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
番茄碱性/中性转化酶SlCIN3基因功能研究;张琼琼;《中国优秀博硕士学位论文全文数据库（硕士）农业科技辑》;20200215;摘要部分，第17页2.2.8节、2.2.10节、2.2.11节、第22页3.2.1节、第32页3.5.1节、3.5.2节、第35页3.5.6节 *

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