CN111235168B

CN111235168B - Receptor kinase SlSRK2B participating in lycopene synthesis and application thereof

Info

Publication number: CN111235168B
Application number: CN202010229657.0A
Authority: CN
Inventors: 周忠静; 邓志平; 谭金娟
Original assignee: Zhejiang Academy of Agricultural Sciences
Current assignee: Zhejiang Academy of Agricultural Sciences
Priority date: 2020-03-27
Filing date: 2020-03-27
Publication date: 2021-04-23
Anticipated expiration: 2040-03-27
Also published as: CN111235168A

Abstract

A receptor kinase SlSRK2B involved in lycopene synthesis and application thereof. The invention relates to the technical field of biology, in particular to a function of a tomato SlSRK2B gene in lycopene synthesis. The nucleotide sequence of the gene SlSRK2B is shown as SEQ ID No: 1, the preparation method comprises the following steps of; and 35S, SlSRK2B, wherein the nucleotide sequence of the used interference fragment is shown as SEQ ID No: 2, the preparation method is as follows. The invention also provides a method for constructing the transgenic tomato, which comprises the following steps: constructing a transgenic tomato by using 35S SlSRK2B RNAi interference vector; the transgenic tomato can improve the content of lycopene and beta-carotene.

Description

Receptor kinase SlSRK2B participating in lycopene synthesis and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a function of a tomato SlSRK2B gene in lycopene synthesis.

Background

Tomato (Solanum lycopersicum L.) is an important economic crop and has high nutritional value. The tomato fruit is accompanied by a series of complex and coordinated physiological and biochemical processes in the maturation process, and is also influenced by some biotic and abiotic stresses. The color of tomato fruit is mainly regulated by lycopene in carotenoid accumulated in large amount in vivo.

The content of the lycopene is one of important indexes for measuring the quality of tomato fruits, the lycopene is a strong antioxidant, has the function of natural pigment, has important functions for preventing a plurality of diseases, and has important significance for researching the synthesis and regulation of the lycopene and improving the quality of the fruits. The synthesis process of lycopene is composed of 5 key enzyme genes: phytoene Synthase (PSY), Phytoene Dehydrogenase (PDS), zeta-carotene dehydrogenase (ZDS), carotenoid isomerase (CRTISO), and Lycopene beta Cyclase (LCY) are accomplished by a series of physiological and biochemical reactions.

SlSRK2B is a Sucrose-non-fermentation Related protein Kinase (Sucrose non-fertilization-1-Related protein Kinase2, SnRK2), belongs to serine/threonine (Ser/Thr) protein Kinase, and SnRK2 is a regulatory factor in abscisic acid (ABA) signal transduction pathway, regulates the mutual connection of multiple signal pathways by modifying target protein through phosphorylation, and plays an important role in various stress reactions such as plant growth and development, pest and disease damage prevention, abiotic stress, ABA signal pathway and the like.

SlSnRK2.6(Solyc08g077780) is a gene with higher homology with SlSRK2B (Solyc01g10394), and according to research reports of Sun et al (Sun et al, 2011) in 2011, the SlSnRK2.6 gene has high expression level in tomato fruit development process and is a key factor of ABA signal pathway.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a gene participating in lycopene synthesis, a protein and application.

In order to solve the technical problems, the invention provides a gene SlSRK2B participating in lycopene synthesis, and the nucleotide sequence of the gene SlSRK2B is shown as SEQ ID No: 1, the preparation method is as follows.

The improvement of the gene SlSRK2B participating in lycopene synthesis provided by the invention comprises the following steps: and 35S, SlSRK2B, wherein the nucleotide sequence of the used interference fragment is shown as SEQ ID No: 2, the preparation method is as follows.

The invention also provides a protein coded by the gene SlSRK2B: the amino acid sequence of the protein is shown as SEQ ID No: 3, respectively.

The invention also provides a plant interference vector containing the gene.

In the present invention, the host cell may be, for example, an E.coli cell or an Agrobacterium cell.

The invention also provides the application of the gene SlSRK2B: is used for increasing the content of lycopene and beta-carotene in tomato fruit. Namely, the gene SlSRK2B is used for constructing transgenic tomatoes, and the transgenic tomatoes can improve the content of lycopene.

The invention also provides a method for constructing the transgenic tomato, which comprises the following steps: constructing a transgenic tomato (namely constructing a tomato SlSRK2B gene silencing transgenic plant) by using 35S SlSRK2B RNAi interference vector; the 35S comprises SlSRK2B, wherein the nucleotide sequence of the interference fragment of the RNAi interference vector is shown in SEQ ID No: 2, the transgenic tomato can improve the content of lycopene and beta-carotene.

The invention constructs a tomato SlSRK2B gene silencing transgenic plant for the first time and performs function research. Through observing the color of tomato fruits of transgenic plants, the SlSRK2B gene is found to play an important role in the biosynthesis process of lycopene. 35S SlSRK2B shows that RNAi transgenic tomato becomes red, so that the content of lycopene is increased, and the gene is involved in lycopene synthesis.

In conclusion, the invention proves that SlSRK2B is a receptor kinase participating in lycopene synthesis by constructing a SlSRK2B gene interference vector and culturing a tomato material with increased lycopene content by a transgenic technology. Has good application prospect in the aspect of improving the content of lycopene in tomato breeding in future. The invention can enrich the synthesis theory of mature pigments of fruits and provide a theoretical basis for improving the content of lycopene by a genetic engineering technology in the future. Has important significance for disclosing the metabolic mechanism of the carotenoid of the tomato fruits and improving the content of the lycopene by utilizing a molecular breeding technology.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is the nucleotide sequence of SlSRK2B gene;

FIG. 2 shows 35S: SlSRK2B: interference fragment in RNAi interference vector;

FIG. 3 is a SlSRK2B protein sequence;

FIG. 4 is 35S: SlSRK2B: RNAi cloning vector map;

FIG. 5 is 35S SlSRK2B RNAi interference vector map;

FIG. 6 is Mico-Tom and 35S: SlSRK2B: RNAi-6 transgenic tomato fruit color;

FIG. 7 shows that SlSRK2B gene silences lycopene content in transgenic plants, namely 35S:SlSRK2B:thecontent of lycopene and beta-carotene in fruits of RNAi-6 transgenic tomatoes in the stage BR +11 (initial extract).

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

firstly, constructing SlSRK2B gene RNAi:

the vector used was pENTR manufactured by Invitrogen corporation^TM/SD/D-

The Cloning Kit, performed as described herein. Through the analysis of the full-length sequence (SEQ ID No: 1) of the SlSRK2B gene, a segment of sequence 169bp (SEQ ID No: 2) is selected as an interference target fragment, and primers are designed as follows:

SlSRK2B::RNAi-L:5’-GGCTCATGAGGAACAAGGAG-3’

SlSRK2B::RNAi-R:5’-ATGGCAAGATGAGTGGGAGT-3’

a sequence fragment (SEQ ID No: 2) is obtained by PCR amplification by taking cDNA of tomato fruits as a template.

25ul of PCR amplification system was used,

high Fidelity enzyme 2x High-Fidelity Master Mix: 12.5ul

SlSRK2B::RNAi-L：1ul

SlSRK2B::RNAi-R：1ul

cDNA template: 1ul

Sterilizing water: 9.5 ul;

the PCR amplification program is 95 ℃ for 5 minutes; 35 cycles: 95 ℃, 45 seconds, 55 ℃, 30 seconds, 72 ℃, 30 seconds; 72 ℃ for 10 minutes.

The gene-specific PCR product is firstly inserted into a cloning vector pENTR, and the map is shown in figure 4; refer to Invitrogen corporation pENTR^TM/SD/

The Cloning Kit, performed as described herein.

Then recombined into an RNAi vector pK7GWIWG2(II) through Gateway LR reaction exchange, as shown in figure 5, the construction method is as follows: LR reaction system: the cloned plasmid pENTR is SlSRK2B:0.5ul, the original plasmid pK7GWIWG2(II)1ul, 1 XTebuffer 1ul, and is placed on ice for 2 minutes, and then 0.5ul of LR reaction enzyme (Proteinase K) is added, and the mixture is left overnight at 25 ℃. The next day, 0.3ul of protease K was added to the LR reaction system at 37 ℃ for 10 minutes, and then the reaction was terminated, and finally all the reaction solution was used for transformation of E.coli DH5 α.

Second, construction and detection of transgenic tomato

Construction of transgenic tomato is slightly modified with reference to Hamza's method (HAMZA and CHUPEAU,1993) as follows:

the constructed binary vector is subjected to agrobacterium infection by tomato (Mico-Tom) cotyledon, dark culture for 2 days, callus induction, resistance induced differentiation and rooting culture to obtain a tissue culture seedling. Transgenic seedlings growing on a culture medium of antibiotic-kanamycin (50mg/L) are screened to obtain RNAi transgenic strains-35S:SlSRK2B:RNAitransgenic strains.

Third, content analysis of lycopene and beta-carotenoid in transgenic tomato

Wild type (Mico-Tom) and 35S:: SlSRK2B:: RNAi transgenic lines tomato plants were started by taking samples of fruit (epicarp) once every two days after the fruit had grown to the broke stage (Break, BR), taking samples of BR +3, BR +5, BR +7, BR +9 and BR +11 (FIG. 6) for a total of 6 stages, taking a certain amount of fruit tissue for grinding, extracting with acetone/n-hexane for 1h, and measuring the content of the anthocyanins in the primary extract by spectrophotometry (NAGATA and YAMASHITA, 1992). As is apparent from FIG. 6, after the color breaking period, 35S: SlSRK2B: fruit color of RNAi transgenic line is obviously red compared with that of control plant, and the fruit shape is different from that of the control plant.

According to 35S SlSRK2B, the content change of lycopene and beta-carotene in RNAi transgenic tomato (figure 7), it can be proved that: the gene SlSRK2B is involved in the biosynthesis of lycopene (or carotenoid).

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Sequence listing

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Met Glu Lys Tyr Glu Leu Val Lys Asp Ile Gly Ser Gly Asn Phe Gly

1 5 10 15

Val Ala Arg Leu Met Arg Asn Lys Glu Thr Lys Glu Leu Val Ala Met

20 25 30

Lys Tyr Ile Glu Arg Gly His Lys Ile Asp Glu Asn Val Ala Arg Glu

35 40 45

Ile Ile Asn His Lys Ser Leu Arg His Pro Asn Ile Ile Arg Phe Lys

50 55 60

Glu Val Val Leu Thr Pro Thr His Leu Ala Ile Val Met Glu Tyr Ala

65 70 75 80

Ala Gly Gly Glu Leu Phe Glu Arg Ile Cys Asn Ala Gly Arg Phe Ser

85 90 95

Glu Asp Glu Ala Arg Tyr Phe Phe Gln Gln Leu Ile Ser Gly Val His

100 105 110

Tyr Cys His Asn Met Gln Ile Cys His Arg Asp Leu Lys Leu Glu Asn

115 120 125

Thr Leu Leu Asp Gly Ser Ala Ala Pro Arg Leu Lys Ile Cys Asp Phe

130 135 140

Gly Tyr Ser Lys Ser Ser Leu Leu His Ser Arg Pro Lys Ser Thr Val

145 150 155 160

Gly Thr Pro Ala Tyr Ile Ala Pro Glu Val Leu Ser Arg Arg Glu Tyr

165 170 175

Asp Gly Lys Leu Ala Asp Val Trp Ser Cys Gly Val Thr Leu Tyr Val

180 185 190

Met Leu Val Gly Ala Tyr Pro Phe Glu Asp Gln Glu Asp Pro Lys Asn

195 200 205

Phe Arg Lys Thr Ile Gln Arg Ile Met Ala Val Gln Tyr Lys Ile Pro

210 215 220

Asp Tyr Val His Ile Ser Gln Asp Cys Arg His Leu Leu Ser Arg Ile

225 230 235 240

Phe Val Ala Asn Ser Ala Arg Arg Ile Thr Ile Lys Glu Ile Lys Ser

245 250 255

His Pro Trp Phe Leu Lys Asn Leu Pro Arg Glu Leu Thr Glu Ala Ala

260 265 270

Gln Ala Ala Tyr Tyr Arg Lys Glu Asn Pro Thr Phe Ser Leu Gln Ser

275 280 285

Val Glu Glu Ile Met Lys Ile Val Glu Glu Ala Lys Thr Pro Pro Pro

290 295 300

Val Ser Arg Ser Val Ser Gly Phe Gly Trp Gly Gly Glu Glu Glu Glu

305 310 315 320

Glu Glu Lys Glu Gly Asp Val Glu Glu Glu Val Glu Glu Glu Asp Asp

325 330 335

Asp Glu Glu Glu Glu Asp Glu Tyr Asp Lys Gln Val Lys Gln Ala His

340 345 350

Gln Ser Leu Gly Glu Val Arg Leu Thr

355 360

Claims

1. GeneSlSRK2BThe use of (a), characterized in that: for increasing the content of lycopene and beta-carotene in tomato fruits;

the geneSlSRK2BThe nucleotide sequence of (A) is shown in SEQ ID No: 1, the preparation method is as follows.

2. The gene according to claim 1SlSRK2BThe use of (a), characterized in that: construction of35S::SlSRK2B::RNAiWhen interfering with the vector, the nucleotide sequence of the interfering fragment used is as shown in SEQ ID No: 2, the preparation method is as follows.