CN107058337B

CN107058337B - Flowering gene CsFT of tea tree and coding protein thereof

Info

Publication number: CN107058337B
Application number: CN201710166191.2A
Authority: CN
Inventors: 郝心愿; 王新超; 杨亚军; 王璐
Original assignee: Tea Research Institute Chinese Academy of Agricultural Sciences
Current assignee: Tea Research Institute Chinese Academy of Agricultural Sciences
Priority date: 2017-03-20
Filing date: 2017-03-20
Publication date: 2020-10-30
Anticipated expiration: 2037-03-20
Also published as: CN107058337A

Abstract

Tea tree flowering geneCsFTAnd the coded protein thereof, belonging to the technical field of molecular biology. The invention provides a tea tree flowering geneCsFTTranscriptCsFTa、TranscriptCsFTbAnd its coded protein, and utilizes said gene to construct plant expression vector, and utilizes the agrobacterium to convert poplar with different genetic backgrounds so as to obtain transgenic poplar plant with obvious early flowering phenomenonCsFTThe plant expression vector of the gene is applied to tea trees, can accelerate the flowering of the tea trees, shorten the growth period of the tea trees and promote the genetic improvement and variety breeding of the tea trees.

Description

Flowering gene CsFT of tea tree and coding protein thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a tea tree flowering geneCsFTAnd the encoded protein thereof.

Background

In the last century, after long-term studies on the mechanism of plant development, it was found that the formation of flower buds of plants was induced by a hormone-like substance produced in leaves. This species is produced in mature leaves and then transported to the apical bud to perform the flower bud-inducing function. The substance for promoting flowering has been intensively studied in the fields of molecular biology and biochemistry in recent years, and as a result, it has been found that the substance for promoting flowering is a substanceFlowering locus T(FT) A protein encoded by the gene. The FT protein has multiple functions in plant growth and developmentThe function is not only as a flowering induction signal with transferability, but also participates in seasonal growth cessation and subsequent dormancy control. Tea is an evergreen woody plant and forms dormant buds in winter to cope with low temperature stress. Moreover, most tea varieties are flowering varieties, and approximately one and a half years elapses from the formation of flower buds to the maturation of seeds. It is seen that, unlike ordinary woody plants, tea plants are undergoing reproductive development throughout the year. Therefore, the growth-dormancy cycle and reproductive development of tea trees are important in the life cycle of tea trees, and the tea production is directly influenced. Meanwhile, partial tea tree resource fruits can be used for extracting oil, are good sources of edible oil and have good health-care function. However, at present, tea plant does not existCsFTThe gene sequence, protein sequence and the related patent report and publication of the function.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to design and provide a tea tree flowering geneCsFTAnd the technical scheme of the coding protein.

The tea tree flowering geneCsFTCharacterized in that the gene has two transcriptsCsFTa and CsFTbTranscript ofCsFTaHas a nucleotide sequence shown as SEQ ID No.1 or a nucleotide sequence which is formed by replacing, deleting or adding one or more nucleotides in the sequence and has the same function, and the transcriptCsFTbHas a nucleotide sequence shown as SEQ ID No.2 or a nucleotide sequence which is formed by replacing, deleting or adding one or more nucleotides in the sequence and has the same function.

The tea tree flowering geneCsFTThe application of the plant early-flowering compound in inducing the early-flowering character of the plant.

The tea tree flowering geneCsFTVectors for transcripts。

The tea tree flowering geneCsFTA host of a vector for the transcript.

The tea tree flowering geneCsFTOf (2) a transcriptCsFTaCharacterized in that the transcriptCsFTaThe nucleotide sequence of (a) is shown as SEQ ID NO: 1 is shown.

The tea tree flowering geneCsFTOf (2) a transcriptCsFTaCharacterized in that the amino acid sequence of the protein is shown as SEQ ID NO: 3, respectively.

The tea tree flowering geneCsFTOf (2) a transcriptCsFTaApplication of protein coded by protein in inducing early flowering characters of plants。

The tea tree flowering geneCsFTOf (2) a transcriptCsFTbCharacterized in that the transcriptCsFTbThe nucleotide sequence of (a) is shown as SEQ ID NO: 2, respectively.

The tea tree flowering geneCsFTOf (2) a transcriptCsFTbCharacterized in that the amino acid sequence of the protein is shown as SEQ ID NO: 4, respectively.

The tea tree flowering geneCsFTOf (2) a transcriptCsFTbAnd the application of the coded protein in inhibiting plant growth arrest and dormant bud formation caused by short sunlight.

The invention provides a tea tree flowering geneCsFTTranscriptCsFTa, transcript CsFTbAnd its coded protein, and utilizes said gene to construct plant expression vector, and utilizes the agrobacterium to convert poplar with different genetic backgrounds so as to obtain transgenic poplar plant with obvious early flowering phenomenonCsFTThe plant expression vector of the gene is applied to tea trees, can accelerate the flowering of the tea trees, shorten the growth period of the tea trees and promote the genetic improvement and variety breeding of the tea trees.

Drawings

FIG. 1 is a drawing ofCsFTaAndCsFTbthe coding region sequence of the gene and the amino acids encoded thereby, are shown in FIG. 1 by the dashed boxesCsFTaAndCsFTbthe 100 th amino acid encoded by the gene.

FIG. 2 is an alignment chart of FT protein sequences in tea trees (CsFTa, CsFTb), Arabidopsis (FT) and poplar (PnFT 1, PnFT 2), and the boxes in FIG. 2 show the tea treesCsFTa/CsFTbThe region of the protein in which amino acid 100 is located, the triangle indicates amino acid 100.

FIG. 3 is a graph showing the sequencing of total cDNA from mature leaves of Camellia sinensis as a templateCsFTGene results plot, triangle generation in FIG. 3Table FT genes open reading frame position of the different bases.

FIG. 4 shows a tea treeCsFTTissue-specific expression profiles of genes.

FIG. 5 shows detection using gene-specific primersCsFTaAndCsFTbexpression profile in axillary buds during dormancy switching of tea.

FIG. 6 shows detection using gene-specific primersCsFTaAndCsFTbpatterns of expression changes in leaves and shoots under dormancy induction.

FIG. 7 shows a tea treeCsFTaGene-overexpressing poplar clone 717 and clone 6 showed early flowering patterns, in FIG. 7A to D are clones 717, E to F are clones 6, D and H are 3-month-old wild-type controls, and from A to C, E to G transformed clones 717 and clone 6 showed strong early flowering after being transferred into rooting medium for 2-3 weeks, with arrows indicating flower-like structures.

FIG. 8 is 5 months oldCsFTbOverexpresses the phenotype of poplar plants under warm, long-day conditions.

FIG. 9 is a schematic view ofCsFTbThe phenotype of the over-expressed poplar plants was characterized under short day conditions, and in FIG. 9, A to D represent clone 717, and E to H represent clone 6. After 3 weeks of short day treatment, the wild type controls (A and E) stopped growing and formed inactive apices, butCsFTbThe top of the over-expressed plants still continued to grow (B and F), and after 9 weeks of short day treatment, the wild type controls had formed intact dormant buds (C and G), but the growing points of the over-expressed plants continued to grow (D and H).

Detailed Description

The present invention is further illustrated by the following examples.

Example 1: tea treeCsFTCloning and sequence analysis of genes

Tea tree was obtained using Longjing 43 as the test materialCsFTThe different transcripts of the gene being obtained by transcriptome sequencingCsFT1050 bp gene is obtained by RACE technology based on gene fragmentsCsFTThe sequence information of the full-length cDNA,CsFTthe full-length cDNA nucleotide sequence is shown as SEQ ID NO: 5, respectively. Open reading frame thereof(ORF) prediction (http:// www.ncbi.nlm.nih.gov/projects/gorf /) shows that the gene is likely to encode a small molecular weight protein of 174 amino acids. Then clone intoCsFTThe nucleotide sequence of the full-length ORF of (1) is shown as SEQ ID NO: 6, and a plurality of clones are picked for sequencing verification, and two kinds of clones are obtainedCsFTTranscripts of the gene, two transcripts differing by only 1 base. However, the difference of 1 base results inFTThe 100 th amino acid encoded by the gene is changed. One is phenylalanine (Phe) and the other is serine (Ser). The two transcripts are named separately asCsFTaAndCsFTb(FIG. 1).CsFTaThe nucleotide sequence of (a) is shown as SEQ ID NO: as shown in figure 1, the first and second main bodies,CsFTathe amino acid sequence of the encoded protein is shown as SEQ ID NO: as shown in figure 3, the first and second,CsFTbthe nucleotide sequence of (a) is shown as SEQ ID NO: as shown in figure 2, the first and second,CsFTbthe amino acid sequence of the encoded protein is asCsFTaSEQ ID NO: 4, respectively.

Then, the tea trees are treatedCsFTThe gene coding amino acid sequence is compared with FT protein sequences in other plants, and the result shows that the cloned tea tree is obtainedCsFTThe gene-encoded protein sequence has high similarity with the FT protein sequence in Arabidopsis and poplar (FIG. 2). Thus clonedFTThe gene should be in Arabidopsis thalianaFTHomologous genes of the gene. At the same time, the results also show that,CsFTthe amino acid sequence conservation of the position of the 100 th amino acid is not high, but the 100 th amino acid is phenylalanine in arabidopsis and poplar. Therefore, in order to further confirmCsFTaAndCsFTbsequence accuracy, using cDNA reverse transcribed from mRNA obtained from mature leaf as template, forCsFTThe gene was re-sequenced and showed two distinct signal peaks at the different bases (FIG. 3). This indicates the presence of teaCsFTaAndCsFTbtwo transcripts.

Example 2: tea treeCsFTAnalysis of Gene expression

First, theCsPTB1Designing universal primer detection for internal reference geneCsFTTissue-specific expression of genes and expression in leaves and axillary buds of different growth states. The results show that it is possible to display,CsFTgene ownerHigh expression in leaves, flowers and shoots, particularly in shoots, is expected to be much higher than in leaves. But the expression in shoots was very low, while there was no expression in stems and roots (FIG. 4). This means thatCsFTThe gene is closely related to flower formation and bud development.

To further understand the two transcripts in teaCsFTaAndCsFTbin order to differentiate the expression patterns of the two genes, we designed specific primers (see Table 1) to distinguish the changes in expression patterns between different dormancy stages and dormancy induction. The results show that, in axillary buds at the para-dormancy (paradormancy), physiological dormancy (endomorphism), ecological dormancy (ecomorphy) and germination stage (germination),CsFTaandCsFTbthere was a high expression level in both the ecological dormancy stage, but there was no significant difference in the expression level between the dormancy-like and ecological dormancy stages (FIG. 5). While in the physiological dormancy and germination phases,CsFTaandCsFTbthere was a clear difference in the expression level of (c). Particularly in the physiological dormancy stage, the expression difference between the two is more obvious. In physiological dormancy and germination stageCsFTbAll the expression levels are obviously higher thanCsFTaExpression of (2).

TABLE 1 primer sequence information

Under the conditions of short sunshine and low temperature dormancy induction,CsFTaandCsFTbthe expression of (2) was significantly suppressed in leaf and apical shoots (FIG. 6). In the leaf and the top shoot,CsFTaandCsFTbthe expression of (a) was significantly elevated at day 1 after the induction of dormancy, and then the expression of both was continuously significantly inhibited. At week 4 after the induction of dormancy, the expression levels of both were lowest. In the blade, withCsFTbIn contrast to the above-mentioned results,CsFTathe expression of (A) is more obviously inhibited by dormancy induction.CsFTaThe expression of (A) was minimal on day 3 of the dormancy induction, andCsFTbthe expression of (a) is gradually decreased, and the expression level is still higher at 4 weeks after the dormancy inductionCsFTa. In the tender shoots at the top end of the plant,CsFTaandCsFTbthere were identical expression changes.

Example 3: functional validation and phenotypic observation

At present, a transformation system of tea trees is immature, poplar as a model plant in woody plants has very comprehensive genetic information, and the dormancy mechanism of the poplar is also researched most thoroughly. Meanwhile, the poplar and the tea tree are both woody plants and have very close genetic backgrounds. Therefore, the function of the gene in the tea tree is most reliable to verify by using the poplar transformation system. The poplar clone 717 and clone 6 selected in the research are general transformation materials in laboratories at present, and have the advantages of easy phenotype discrimination, clear genetic background and high transformation rate. In addition, it was found that dormancy (growth arrest and dormant bud formation) of poplar was affected only by photoperiod, independent of temperature (Resman et al 2010). Therefore, the dormancy induction conditions can be well controlled, and the influence of environmental factors on the tea tree genes when the tea tree genes play a role in the poplar can be known.

CsFTaAndCsFTbthe overexpression vectors of (1) are respectively transferred into a poplar clone 717 and a poplar clone 6, and corresponding plants transferred into empty vectors are respectively used as wild type controls. The positive plants successfully screened by the transformation system are screened by hygromycin resistance genes and expression screening. Multiple independently transformed plants that have been verified to have been successfully transformed and to express the gene of interest with high efficiency are used for subsequent phenotypic observation and experimental treatment.

Overexpression in PopulusCsFTaAndCsFTb. Results displayCsFTaThe over-expressed poplar plants showed strong early flowering promoting phenomenon. Generally, it takes ten years for poplars to bloom, andCsFTathe over-expressed plants started to form obvious flower-like structures 2-3 weeks after being transferred into rooting induction medium. In clone 717, some of the explants formed shoots forming a radial floral structure directly at the tip (FIG. 7A), and some formed buds or butterfly receptacle structures at the tip (FIGS. 7B and C). In clone 6, a floral structure also appeared (fig. 7E, F, G). At the same timeCsFTaThe over-expressed plants do not root after being transferred into the rooting induction medium, but flower directly.And shoots or plants after flowering soon senesced and died (fig. 7E, F). It can be seen thatCsFTOverexpression of the gene continuously promotes flowering and has affected the normal development of plants. At the same time, the flower-like structure formed by the transformed plant is not a complete flower organ, which also indicates that the normal formation of the flower is not the same as that of the plantFTBesides genes, other genes need to be involved together. D and H in fig. 7 are wild type controls for clone 717 and clone 6, respectively. The control plants were 3 months old, grew normally and showed no premature flowering.

But under the same conditions, however,CsFTbthe over-expressed poplar plants did not show early flowering. The transformed plants showed the same growth and performance as the wild type control. FIG. 8 shows a 5 month oldCsFTbOverexpresses the phenotype of poplar plants and wild-type controls. To identifyCsFTbWhether the overexpression plants affected the dormancy formation of the plants or not, and the transformed plants of 5 months old were transferred to a short-day dormancy induction incubator and a long-day low-temperature incubator for treatment, the results showed that the wild type control stopped growing after 3 weeks of treatment under the short-day treatment conditions, and the apical growing point began to form a resident bud (FIG. 9). WhileCsFTbThe over-expressed plants are affected by short sunlight and show a state of continued growth. After 9 weeks of short day induction, the control plants had completely ceased growing, the top leaves had begun to senesce, and full and scale-covered dormant buds had formed. Whereas the over-expressed plants continued to grow, especially clone 717 did not show growth arrest or formation of dormant buds. Clone 6 grew slowly, but the growth point was still active. The over-expressed plants transferred into the long-day low-temperature culture condition showed no phenotypic difference compared with the control. However, the growth of the poplar is directly influenced by the low temperature, and the growth of the test plant is quickly inhibited under the condition that the test plant is transferred to the low temperature. Since the dormancy of poplar is affected only by the light cycle, the plant under the low temperature condition has no dormant bud.

Thus, over-expression of tea plantCsFTaCan obviously promote flowering, and tea treeCsFTbThere is no phenomenon of promoting early flowering. But will beCsFTbAfter short-day treatment of the over-expressed plants, the over-expression was compared with the controlThe plant can obviously inhibit the growth arrest and the formation of dormant buds caused by short sunlight.

SEQUENCE LISTING

<110> institute of tea leaf of Chinese academy of agricultural sciences

<120> tea tree flowering gene CsFT and encoding protein thereof

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atgcctcggg acagggatcc tcttgttgtt gggcgtgtta taggggatgt tttggacccc 60

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gagctcaggc cttctcaagt tgtcatccaa cctagggttg acattggtgg tgatgacctt 180

aggaccttct acactcttgt tatggtggac cctgatgctc ccagcccaag tgacccaaac 240

ctaagggaat acttgcattg gttggtgact gatatcccag caaccactgg agcaagcttt 300

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Claims

1. Tea tree flowering geneCsFTOf (2) a transcriptCsFTbAnd the application of the coded protein in inhibiting the growth arrest and dormant bud formation of plants caused by short sunlight, and the transcriptCsFTbThe nucleotide sequence of (a) is shown as SEQ ID NO: 2, said transcriptCsFTbThe amino acid sequence of the encoded protein of (1) is shown in SEQ ID NO: 4, respectively.