CN116024252B - Use of SlTCP gene or related biological material in improving tomato character - Google Patents
Use of SlTCP gene or related biological material in improving tomato character Download PDFInfo
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
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Abstract
The invention belongs to the field of biotechnology and genetic breeding, and particularly relates to application of SlTCP gene or related biological materials thereof in improving tomato traits. The invention discloses the regulation and control effect of SlTCP on tomato plant height, leaf dry matter weight and chlorophyll content by cloning tomato TCP transcription factor SlTCP and constructing over-expression transgene material. The TCP transcription factor family member can be applied to regulation and control of tomato plant type and yield, and provides an effective way for solving the fruit quality of fruits and vegetables such as tomatoes.
Description
Technical Field
The invention belongs to the field of biotechnology and genetic breeding, and particularly relates to application of SlTCP gene or related biological materials thereof in improving tomato traits.
Background
Tomatoes are a typical glaring plant as the main facility cultivated crop in china. The current research on tomato quality and yield is mainly focused on tomato fruits, and less research on leaves is performed. Leaves are important organs of plants, and the plants can provide continuous power for the growth and development of the plants through photosynthesis and other functions. In the evolution process of plants, the leaves of the plants are quite different, and the leaves of different plants are different in shape, size and even color; leaves of the same plant at different development periods are different. The chlorophyll content of tomato leaves affects photosynthesis and starch accumulation of tomatoes, which in turn affects fruit development and fruit quality of tomatoes.
Leaf color is mainly affected by chlorophyll, carotenoid and other chemical substances in tissues, and leaves with normal photosynthesis mostly show green color, but leaf color is also greatly different in different environments. The mutation of the Os PL6 gene can cause anthocyanin accumulation in rice, and leaves are purple, so that photosynthetic efficiency of the rice leaves is reduced. In tomato, silencing BR receptor SlBRI1 can result in tomato leaf curl and dark green. The transcription factor can be used as a binding protein of a gene promoter region, and can also regulate chlorophyll content in plants, for example, the GLK2 gene can be overexpressed in tomatoes to increase pigment content.
TCPs are a class of plant-specific transcription regulatory factors that play an important role in plant development. In 1999, cubas et al reported the TCP gene family for the first time. AtTCP14 and AtTCP of arabidopsis regulate internode length and leaf shape; BAD1 in corn can regulate the angle of corn lateral branch differentiation from main stem by promoting cell proliferation in leaf pillow, thereby affecting corn plant type. The molecular pathway of TCP protein action and its related studies on the mode of interaction with DNA have made great progress, but the action in tomato has not been reported yet.
Disclosure of Invention
One of the purposes of the present invention is to provide the use of SlTCP gene or its related biological material in improving tomato trait, said biological material is an expression cassette, recombinant vector, recombinant bacterium or transgenic cell line containing said SlTCP gene;
The traits include plant height, leaf dry matter weight and chlorophyll content;
the sequence of SlTCP gene is shown as SEQ ID NO. 1.
Further, the SlTCP gene has increased activity and/or expression level in tomato, and the tomato has increased plant height, leaf dry matter weight and chlorophyll content.
The second object of the invention is to provide a method for improving tomato traits, which comprises up-regulating the expression of SlTCP gene, so that the tomato plant height, the dry matter weight of leaves and the chlorophyll content are improved, and the sequence of SlTCP gene is shown as SEQ ID NO. 1.
Further, up-regulating the expression of SlTCP gene includes: the SlTCP gene or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the gene are transferred into tomato.
It is a further object of the present invention to provide a method for the directed selection or identification of tomatoes with superior traits, said method comprising: identifying and testing the expression of SlTCP gene in tomato, wherein the sequence of SlTCP gene is shown as SEQ ID NO. 1;
If SlTCP gene expression of the tested tomatoes is obviously higher than the average expression value of the tomatoes, the tomatoes with excellent properties are expressed as plant height, leaf dry matter weight and chlorophyll content are improved.
Further, the expression of SlTCP gene in the test tomatoes was identified by fluorescent quantitative PCR.
Furthermore, the sequence of the primer identified by the fluorescent quantitative PCR method is shown as SEQ ID NO. 4-5.
The invention has the following beneficial effects:
The invention discloses an application of tomato TCP transcription factor family members in regulating and controlling growth and development of tomatoes. The application provided by the invention is the application of SlTCP gene to increase tomato plant height, leaf dry matter weight and chlorophyll content. The invention utilizes the vector for guiding the expression of exogenous genes in plants to introduce SlTCP gene into tomato cells for over-expression, and can improve the plant height, leaf dry matter weight and chlorophyll content of tomatoes. The invention has important theoretical and practical significance for researching molecular mechanism of high yield of tomatoes and breeding high-quality varieties.
Drawings
FIG. 1 is a gel electrophoresis diagram of a PCR product of a bacterial liquid of Escherichia coli of pBWA (V) HS-SlTCP14, M: DL2000DNA MARKER;1-4, the target fragment.
FIG. 2 is the acquisition of tomato transgenic plants, A: aseptic seedlings; b: co-culturing; c: cultivating buds; d: rooting culture; e: hardening seedlings; f: transplanting.
Fig. 3 shows the expression level analysis of SlTCP over-expressed plant leaves, showing significant level of difference (P < 0.05) and very significant level of difference (P < 0.01) compared to normal AC tomato leaves
FIG. 4 shows comparison (A) and quantitative statistics (B) of tomato plant height development for AC and OE-SlTCP 14.
Fig. 5 is SlTCP showing that the chlorophyll content of the plants overexpressed by SlTCP shows that the difference reaches significant level (P < 0.05) and shows that the difference is extremely significant level (P < 0.01) compared to normal AC tomato leaves.
Fig. 6 is AC and OE-SlTCP14 tomato dry matter weights, indicating significant levels of difference (P < 0.05) compared to normal AC tomato leaves, indicating very significant levels of difference (P < 0.01).
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
Example 1: construction of the overexpression vector pBWA (V) HS-SlTCP14
Extracting tomato RNA, reversely transcribing cDNA, and amplifying SlTCP target fragment (sequence shown as SEQ ID NO. 1) by using cDNA as a template; the target fragment was ligated with the linearized pBWA (V) HS vector (cohesive end Bsa I/Eco 31I) to obtain recombinant plasmid pBWA (V) HS-SlTCP14. Then, the escherichia coli is transformed, monoclonal escherichia coli bacterial liquids are screened for PCR amplification identification (as shown in figure 1), and the bacterial liquids are amplified to form bands with the same size as the target gene (798 bp). Then sequencing, extracting plasmids from the bacterial liquid with correct sequencing, and transferring the recombinant plasmids into agrobacterium competent GV3101 by adopting a liquid nitrogen freeze thawing method for genetic transformation of tomatoes.
The primer sequences for amplifying the SlTCP target fragment are as follows:
SlTCP14-F(SEQ ID NO.2):5'-ATGTCGACGTCTGCAGA-3'；
SlTCP14-R(SEQ ID NO.3):5'-ACGTCCGTCGTCGTC-3'。
Example 2: genetic transformation of tomato
The recombinant vector plasmid was transformed into tomato by leaf disc method using AC tomato (hereinafter or abbreviated as AC) as a test material to obtain a stable genetic transformation material. When tomato seedlings in the culture medium grow until cotyledons are fully stretched (figure 2A), cutting cotyledons with the length of 0.5cm, placing the cotyledons in a preculture medium, carrying out dark culture for 2d, enabling agrobacterium to infect the tomato cotyledons, placing the back of the infected cotyledons in a co-culture medium, carrying out dark culture for 2d, transferring the tomato seedlings into a germination culture medium for culture (figure 2B), and then replacing the culture medium at intervals of two weeks. When adventitious buds grow out, they are transferred to a bud-growing bottle (FIG. 2C), and when adventitious buds grow to 2-3cm, adventitious buds are cut from the base of the stem and placed in rooting medium for culture (FIG. 2D). After adventitious buds root, taking out the complete plant from a culture bottle, cleaning the culture medium, placing the complete plant into a kawasaki nutrient solution for hardening seedlings (figure 2E), transplanting the complete plant into a nutrition pot for culturing after the plant is suitable for the external environment, and obtaining the transgenic tomato plant with the over-expression SlTCP14 (figure 2F).
Example 3: analysis of SlTCP.sup.14 expression level in transgenic tomato leaves
To determine SlTCP expression levels, RNA was extracted and reverse transcribed into cDNA using normal AC tomato leaf as a control. qRT-PCR analysis was performed using the SlTCP real-time quantitative primer (F (SEQ ID NO. 4): TCGACGTCTGCAGAGGG; R (SEQ ID NO. 5): CTGAAAAACTCTTGCCGCA)) of 14 with the action gene as an internal reference.
As shown in FIG. 3, the expression levels in the leaves of the over-expressed transgenic lines were significantly increased compared to AC, indicating that the over-expression vector pBWA (V) HS-SlTCP14 had been successfully transformed into AC tomato. The expression level of the strain No.4 and No. 17 and No. 18 is obviously higher than that of the AC tomatoes by 14-17 times, and other strains are also improved by about 5 times. Therefore, three transgenic lines with high over-expression efficiency, namely No.4, no. 17 and No. 18, are selected for subsequent experiments.
Example 4: determination of the Properties of the overexpressed transgenic tomato
1. Plant height measurement
By preliminary analysis of the phenotype of the three selected transgenic lines. The transgenic lines were higher in height compared to the wild type (fig. 4, a), and by measuring the 3-strain height, the transgenic lines were found to be significantly higher than AC tomatoes (fig. 4, b).
2. Determination of chlorophyll content in leaves
0.2G of the leaf blade is weighed and placed into a 50ml centrifuge tube, 25ml of ethanol solution with concentration of 95% is added into the centrifuge tube in advance, and the centrifuge tube is placed at a place where the leaf blade is protected from light for overnight soaking until the leaf blade is completely green-removed and decolored. The absorbance of the solution was measured with an ultraviolet spectrophotometer at 470nm,649nm and 665nm, respectively, using a 95% ethanol solution as a control. The chlorophyll content was calculated according to the following formula:
Ca＝13.95*D665-6.88*D649
Cb＝24.96*D649-7.32*D665
chlorophyll content (mg/g) =pigment concentration C extract volume (L)/fresh sample weight
Chlorophyll content was found to be significantly higher than that of AC tomatoes by measuring the chlorophyll content of the T0 transgenic lines (fig. 5).
3. Dry matter determination
To verify whether an increase in chlorophyll content has an effect on leaf dry matter weight. 1.5g of fresh leaves of each of three transgenic strains AC tomato, OE4, OE17 and OE18 are weighed, the leaves are deactivated for 30 minutes at 105 ℃, and the leaves are dried in an oven at 65 ℃ for 5 hours, and the weight of the dry matters is measured. As shown in fig. 6, the leaf dry weight of the transgenic lines was significantly higher than AC tomatoes.
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. It is therefore intended that the following claims be interpreted as including the 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 modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (6)
- The application of the SlTCP14 gene or biological material thereof in improving tomato traits is characterized in that the biological material is an expression cassette, a recombinant vector or a recombinant bacterium containing the SlTCP gene;the characteristics are plant height, dry matter weight of the leaves and chlorophyll content;The sequence of SlTCP gene is shown as SEQ ID NO. 1;And improving the expression quantity of SlTCP gene in tomato, so as to improve the plant height, leaf dry matter weight and chlorophyll content of tomato.
- 2. A method for improving tomato characters is characterized in that the expression of SlTCP gene is up-regulated, so that the tomato plant height, the dry matter weight of leaves and the chlorophyll content are improved, and the sequence of SlTCP gene is shown as SEQ ID NO. 1.
- 3. The method of claim 2, wherein up-regulating the expression of SlTCP gene is by transferring said SlTCP gene or an expression cassette, recombinant vector or recombinant strain containing said gene into tomato.
- 4. A method for directionally selecting or identifying tomatoes with good traits, comprising: identifying and testing the expression of SlTCP gene in tomato, wherein the sequence of SlTCP gene is shown as SEQ ID NO. 1;If SlTCP gene expression of the tested tomatoes is obviously higher than the average expression value of the tomatoes, the tomatoes with excellent properties are expressed as plant height, leaf dry matter weight and chlorophyll content are improved.
- 5. The method of claim 4, wherein the expression of SlTCP gene in the test tomato is identified by fluorescent quantitative PCR.
- 6. The method of claim 5, wherein the sequence of the primers used for the fluorescent quantitative PCR assay is set forth in SEQ ID NO. 4-5.
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| CN101969759A (en) * | 2007-12-20 | 2011-02-09 | 巴斯夫植物科学有限公司 | Plants having enhanced yield-related traits and a method for making the same |
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| CN110042109A (en) * | 2019-05-14 | 2019-07-23 | 长江师范学院 | Gene relevant to tomato leaf aging and its application |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2016094547A1 (en) * | 2014-12-09 | 2016-06-16 | Texas Tech University System | Transcription factors and method for increased fiber length of cotton |
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| CN109628439A (en) * | 2018-12-11 | 2019-04-16 | 沈阳农业大学 | A kind of gene and application promoting tomato Chlorophyll synthesis and photosynthetic efficiency |
| CN110042109A (en) * | 2019-05-14 | 2019-07-23 | 长江师范学院 | Gene relevant to tomato leaf aging and its application |
| 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 |
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| Title |
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