CN111471708A

CN111471708A - Method for changing stem node characters of plants and method for constructing transgenic plants

Info

Publication number: CN111471708A
Application number: CN202010391661.7A
Authority: CN
Inventors: 王树伟; 肖云平; 史贤俊; 王翠亭
Original assignee: Shanghai Oe Biotech Co ltd
Current assignee: Shanghai Oe Biotech Co ltd
Priority date: 2020-05-11
Filing date: 2020-05-11
Publication date: 2020-07-31

Abstract

The invention belongs to the field of construction of transgenic plants, and relates to a method for changing the stem node character of a plant and a method for constructing a transgenic plant. The PtCYP714F1 gene sequence provided by the invention can change the phenotypic character of plant stems, so that the length of internodes of a transgenic plant is shortened, the number of nodes is increased, and the number of axillary buds or leaves is increased. The invention can improve the stem node characters of various plants by utilizing the PtCYP714F1 gene sequence, thereby cultivating more excellent plant varieties and having very high application value in the development and cultivation of new plant varieties.

Description

Method for changing stem node characters of plants and method for constructing transgenic plants

Technical Field

The invention belongs to the field of construction of transgenic plants, and relates to a method for changing the stem node character of a plant and a method for constructing a transgenic plant, wherein a PtCYP714F1 gene derived from poplar is transferred into the plant. The method has high potential application value in the aspect of improving the plant variety characters, and provides a very valuable gene resource for cultivating new plant varieties by using molecular breeding technologies such as transgenosis and the like.

Background

The stem of the plant plays a role in supporting the plant, and is also responsible for transporting water, inorganic salts and the like absorbed by the root to each part of the plant body, thereby playing a vital role in the growth and development of the plant. The plant type of the plant relates to various characters such as plant height, stalk character, node number of main stems, internode length, branch number and the like. Although the number of nodes in the main stem of a plant has been considered to be a complex quantitative trait, the number of nodes and internode length of the main stem are relatively fixed and can be stably inherited for a particular plant variety.

Plant height is one of the important traits of plants, and the key factor determining the plant height trait of plants is the height of stems. The stem is composed of internodes connected by nodes. The nodes on the plant stem are the implantation parts of the buds and the leaves, and the part between two adjacent nodes is called internode. Thus, the length of the internodes and the number of nodes determine the height (length) of the stem.

Gibberellins (GA) are the major plant hormones that control plant height phenotype. The synthesis and metabolism of gibberellin involve the participation of various enzymes, and at least ten related genes have been reported, and most of the genes belong to the P450 family. Among the reported genes, most of the genes (such as OsCYP714D1, AtCYP714A1 and AtCYP714A2) of CYP714 family derived from various plants such as rice, Arabidopsis, poplar and the like have similar functions in the aspect of regulating plant height, namely, when the genes are over-expressed in transgenic plants (rice and Arabidopsis), the content of active GA molecules in the plants is reduced by inactivating the active GA molecules in a non-13-position hydroxylation synthesis pathway, so that internodes of the transgenic plants are shortened, and finally the plant height of the transgenic plants is shortened.

However, to date, there has been no report that any gene, including the CYP714 family gene, has a function of changing the number of plant nodes.

Disclosure of Invention

Through the function research of the PtCYP714F1 gene sequence, the invention provides that the gene sequence not only has the function similar to other CYP714 family genes (shortens plant internodes), but also has the new function of increasing the number of stem nodes and the corresponding internode number and increasing the number of axillary buds or leaves. Therefore, the invention provides a method for simultaneously changing the number of plant stem nodes and internode length by transferring the PtCYP714F1 gene sequence into different plants. By transferring PtCYP714F1 into different plants, such as gramineae plants like rice, bamboo, sugarcane, etc., vegetable plants, or some ornamental plants with ornamental stems or ornamental leaves, the characteristics of the stems of the plants can be improved, thereby realizing the development and utilization of related new varieties.

The invention provides a PtCYP714F1 gene sequence cloned from poplar, as shown in SEQ ID No.1, the gene sequence has the function of changing the phenotypic character of plant stems, and comprises the steps of increasing the number of stem nodes and the corresponding internode number, shortening the internode length and increasing the number of axillary buds or leaves.

The invention uses the transgenic technology to transfer the PtCYP714F1 gene sequence from poplar into other plants (such as rice), which can increase the number of stem nodes and the corresponding internode number of the transgenic plants, shorten the internode length and increase the number of axillary buds or leaves.

The invention utilizes transgenic technology to transfer the PtCYP714F1 gene sequence from poplar into other plants (such as rice), and can increase the number of axillary buds or leaves by increasing the number of stem nodes of transgenic plants.

According to the invention, the excessive expression of the PtCYP714F1 gene is adopted, so that the internode length is shortened while the number of transgenic plant nodes is increased.

The invention provides a method for changing the stem node character of a plant and/or changing the plant type of the plant and/or improving the yield or biomass of the plant, which comprises the steps of transferring a PtCYP714F1 gene sequence into a plant cell by a transgenic technology; alternatively, the method comprises increasing expression of a PtCYP714F1 gene sequence in the plant.

Wherein the method comprises increasing the number of stem nodes, and/or increasing the number of internodes, and/or increasing the number of axillary buds or leaves of the transgenic plant.

The invention provides a method for constructing a transgenic plant, which comprises the step of transferring a PtCYP714F1 gene sequence into a plant cell by a transgenic technology.

The invention also provides a plant cell obtained by adopting the method for transformation.

The invention also provides a method for constructing transgenic rice, which comprises the following steps:

(1) b L AST comparison is utilized to find out a gene sequence homologous with rice OsCYP714D1 from a sequenced poplar variety, a primer is designed, and a PtCYP714F1 gene is obtained by amplification from the sequenced poplar variety, and sequencing verification is carried out (SEQ ID No. 1).

(2) A promoter proD1 of an OsCYP714D1 gene is cloned from rice, a plant expression vector is constructed, the PtCYP714F1 gene is transferred into rice callus, and a plurality of resistant strains are obtained by inducing callus differentiation and multi-round hygromycin screening.

(3) And performing PCR identification on the screened resistant strain, and determining the PCR positive strain as the PtCYP714F1 transgenic strain.

(4) And performing RT-PCR detection on the expression level of the PtCYP714F1 gene in the transgenic line, and determining the expression quantity of the PtCYP714F1 gene in different transgenic lines.

(5) Phenotypic analysis and comparison of transgenic lines and controls (eui mutant and wild type) were performed, and internode length and number of stem nodes were counted.

The invention also provides a transgenic plant cell obtained by the method.

The invention also provides a vector, which comprises the PtCYP714F1 gene sequence.

Preferably, the vector further comprises one or more control sequences capable of driving expression of the PtCYP714F1 gene sequence; and optionally transcribing the termination sequence.

The invention also provides a plant cell comprising the PtCYP714F1 gene sequence as described above.

In the present invention, homologous genes having homology of 80% or more, 85% or more, 88% or more, and 90% or more (including 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) with the PtCYP714F1 gene (SEQ ID No.1) are also within the scope of the present invention, which can achieve the above-described functions of the present invention.

In the present invention, the homology with the PtCYP714F1 gene (SEQ ID No.1) is 80% or more, 85% or more, 88% or more, or 90% or more, and the homologous gene capable of realizing the above-mentioned functions of the present invention is also within the scope of the present invention.

The invention has the beneficial effects that: the PtCYP714F1 gene sequence provided by the invention can change the phenotypic character of plant stems, so that the length of internodes of a transgenic plant is shortened, the number of nodes is increased, and the number of axillary buds or leaves is increased. The PtCYP714F1 gene sequence can be used for improving the stem node characters of gramineous plants, vegetable crops and some foliage plants on one hand, so as to cultivate more excellent plant varieties (for example, the plant can change the water or mineral absorption, transportation and utilization capacity, so as to play a role in saving water and resisting drought), and on the other hand, the plant type can be changed, so that the yield or biomass (such as vegetables, bamboos, sugarcane and the like) can be improved, and the ornamental property (such as landscape or foliage plants, flowers and the like) of the plants can be increased. Therefore, the method has very high application value in the development and cultivation of new plant varieties.

Drawings

FIG. 1 shows the identification and overexpression detection of PtCYP714F1 transgene. And A, PCR identification. M, DNA marker; WT, wild type; h₂O, negative control; f1-1, F1-15-F1-17, F1-19-F1-25, different resistant strains. RT-PCR molecular identification: and (3) carrying out overexpression analysis on the PtCYP714F1 gene in the transgenic rice line. WT, wild type; f1-1, F1-16-F1-17, F1-19-F1-24, different transgenic rice lines.

FIG. 2 shows the comparison of the PtCYP714F1 transgenic line and the phenotype of the control group (wild type WT and its mutant eui). A. And comparing the plant height phenotype. B. The major and internodal phenotypes, the location of the nodes are indicated by arrows. C. Major internode length phenotypic comparisons.

FIG. 3 is a comparison of internode numbers between the PtCYP714F1 transgenic line and the control group (wild type WT and its mutant eui).

FIG. 4 shows the comparison of the PtCYP714E4 transgenic lines with the control (wild type WT and its mutant eui) phenotype. a. And comparing the plant height phenotype. b. The major and internodal phenotypes, the location of the nodes are indicated by arrows.

FIG. 5 shows the comparison of the PtCYP714E5 transgenic lines with the control (wild type WT and its mutant eui) phenotype. a. And comparing the plant height phenotype. b. The major and internodal phenotypes, the location of the nodes are indicated by arrows.

Detailed Description

The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.

Example 1 cloning of PtCYP714F1 Gene

5'-atggaagggttccccatctccta-3' (SEQ ID NO.3) and 5'-tgtccaccctcaggaatcatatctt-3' (SEQ ID NO.4) are used as primers, cDNA (https:// mycocosm. jgi. doe. gov/Poptr1_1/Poptr1_1.home. html) after reverse transcription of total RNA of a sequenced poplar variety is used as a template, a specific band of about 1.5kb is amplified, and the specific band is proved to be a PtCYP714F1 gene through sequencing verification and B L AST comparison, and the CDS total length of the gene is 1464bp (SEQ ID NO. 1).

Example 2 construction of the p1301proD1-PtCYP714F1 transformation vector

First with 5-ctgcagtcacgtacacgcatgcccacct-3' (SEQ ID NO.5) and 5-agatctcctttctctctcatccgacggt-3' (SEQ ID NO.6) is used as a primer, a promoter proD1(SEQ ID NO.2) of the rice OsCYP714D1 gene is cloned from rice genome DNA, and the promoter proD1 sequence is connected to the PstI/BglII locus of a pCAMBIA1301 vector (see http:// www.bios.net/day/cambia/585. html) to construct an intermediate vector p1301proD 1. Then, the PtCYP714F1 gene obtained in the embodiment 1 of the invention is connected into BamHI/PmlI locus of an intermediate vector p1301proD1 to construct a plant expression vector p1301proD1-PtCYP714F1 for over-expressing the PtCYP714F1 gene in rice. Agrobacterium tumefaciens (strain EHA105, see Hood, E.E.et al, Transgenic Res.,1993,2,208-218) mediated transformation of a mutant eui of rice variety ZH11 (the eui mutant differs from the wild type in phenotype in that the uppermost internode of the eui mutant rice is elongated).

Example 3 genetic transformation of Rice

(1) Induction of rice mature embryo callus: rice seeds are hulled, soaked in 70% ethanol for 1min, soaked in 20% (v/v) sodium hypochlorite solution for 20min (continuously shaken during the soaking period), and then fully washed with sterile water for 3-5 times. Seeding on NB callus induction culture medium after sterile filter paper is sucked dry, performing dark culture at 26-28 ℃ for 4 weeks, and inoculating callus particles on new NB callus induction culture medium for dark culture for 4 days for later use;

(2) agrobacterium containing the p1301proD1-PtCYP714A3 plasmid was inoculated in L B medium (containing Kan 50ug/ml, Rif 25ug/ml) the day before transformation, and shake-cultured at 28 ℃ and 200rpm (overnight) until OD66o was 0.6-0.8;

(3) transferring the callus into a sterile triangular flask, and pouring the cultured agrobacterium liquid (so that the callus is immersed by the liquid);

(4) standing at room temperature for 20min, and gently shaking for several times;

(5) pouring out the bacterial liquid, transferring the callus to sterile filter paper to absorb excess bacterial liquid, transferring to NB co-culture medium, and performing co-culture at 20-25 ℃ in the dark for 2-3 days;

(6) transferring the co-cultured callus into an aseptic triangular flask, washing with sterile water for 2-3 times, and washing with sterile water containing 500 ml/L carbenicillin for 20 min;

(7) after the excess water is sucked off on the sterile filter paper, transferring the callus to an NB screening medium (containing 200 mg/L timentin and 50 mg/L hygromycin) for screening of transformed cells, wherein 3 weeks is a period, and the screening is carried out for 2-3 periods;

(8) transferring the screened resistant callus to a pre-differentiation culture medium for culturing for 1 week, and then transferring to an NB differentiation culture medium (containing BAP 2 mg/L and NAA 0.5 mg/L) for culturing under the conditions of 26 ℃, 16h of light/8 h of darkness;

(9) transferring the differentiated resistant regeneration plant to a rooting culture medium (containing 1/2MS and NAA 0.5 mg/L) to strengthen the seedling and root;

(10) after about 3 weeks, rooted, regenerated resistant plants are transplanted into the greenhouse.

Example 4 PCR identification and RT-PCR detection of transgenic Rice

More than 20 hygromycin-screened and transplanted to survive resistant strains, randomly selecting 11 strains from the strains to extract leaf DNA, and carrying out PCR identification by taking 5'-TCTGGTGGAGGCCCGAGCTAA-3' (SEQ ID NO.7) and 5'-AAGCAGGTTTTCCCAGGTCCA-3' (SEQ ID NO.8) as primers. 9 of the 11 resistant strains were transgenic positive strains (FIG. 1A) numbered F1-16-F1-17, F1-19-F1-24 and F1-1.

Extracting RNA of wild rice and transgenic rice, performing reverse transcription to obtain cDNA, and performing RT-PCR molecular identification with the same primers. The results show that the PtCYP714F1 gene in the transgenic line can be over-expressed, and although the expression level is different among different lines, the transgenic line with the over-expressed PtCYP714F1 gene shows increased internode number (figure 1B), wherein the difference of the expression level is usually related to the insertion site of the transgene and the copy number integrated on the genome.

Example 5 phenotypic analysis of transgenic Rice

Phenotypic observation and statistics of transgenic rice lines and control groups (wild-type WT and eui mutants thereof) revealed that the plant type of PtCYP714F1 transgenic line was significantly shorter in plant height than the eui mutant, but was generally higher than the wild-type (fig. 2A). After the leaves covering the stem were peeled off, the PtCYP714F1 transgenic strain was found to be significantly different in phenotype of the stem from the control group (fig. 2B, C), including: (1) the uppermost internode is shortened, which indicates that the PtCYP714F1 gene has similar functions with other reported genes in the CYP714 family; the other internodes below the uppermost internode are shortened to different degrees, which shows that the PtCYP714F1 gene has a dwarfing function on the uppermost internode and also has a certain dwarfing function on other internodes; (2) the number of nodes of the stem is remarkably increased, the function is firstly discovered and verified by the invention, the number of the nodes of the control group and a plurality of transgenic lines obtained by the invention is counted in the current report that no other genes have similar functions, and the result shows that the transgenic lines generally exist in 5-7 internodes at the positions corresponding to 3 internode positions from the top to the base of the control lines (WT and eui) which are remarkably more than the control lines (WT and eui) (figure 3).

The above results indicate that PtCYP714F1 has a function of changing the number of plant nodes and corresponding internodes in addition to the plant height-regulating trait by GA pathway common to other CYP714 families, which has not been reported before in the present invention.

According to the existing literature reports, the CYP714 family genes (including rice-derived OsCYP714D1 (ref: Zhu Y et al, 2006, Plant Cell 18(2): 442- & 456), OsCYP714B1/B2 (ref: Magome H et al, 2013, PNAS 110(5): 1947-1952), and Arabidopsis-derived AtCYP714A1/A2 (ref: Zhang Y et al, 2011, the Plant Journal 67(2): 342- & 353)) in rice and Arabidopsis were unable to change the node and internode numbers of transgenic plants, and in the previous studies of the present invention, it was also confirmed that the poplar-derived PtCYP714A3 gene did not have the function of changing the stem node and the corresponding internode numbers of transgenic plants (ref: Wang et al, 2016, Plant technology Journal14(9): 1838-1851).

In order to determine whether only PtCYP714F1 in CYP714 family has the function of changing the stem node and internode number of a plant, the invention respectively verifies another 2 genes-PtCYP 714E4 and PtCYP714E5, which are also derived from poplar CYP714 family, by using the same vector construction and transgenic method, and by phenotypic observation, the invention verifies that the 2 genes also have the function of changing the plant height phenotype shared by other CYP714 family genes, but do not change the stem node and the corresponding internode number (figure 4 and figure 5).

The combination of the above results can clearly conclude that: the PtCYP714F1 gene (SEQ ID No.1) has the function of changing the number of plant stem nodes and the corresponding internode number and increasing the number of axillary buds or leaves, which is a specific function of the gene, and other known homologous genes do not have the function.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected.

SEQUENCE LISTING

<110> Shanghai Ouyi biomedical science and technology Limited

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Claims

Use of the sequence of the PtCYP714F1 gene to increase the number of stem nodes, and/or increase the number of internodes, and/or increase the number of axillary buds or leaves of a transgenic plant.
2. The use of claim 1 or 2, wherein the PtCYP714F1 gene sequence is also used to shorten internode length.
3. The use according to claim 1 or 2, wherein the PtCYP714F1 gene sequence is set forth in SEQ ID No. 1; or the PtCYP714F1 gene sequence has homology of more than 80 percent, or more than 85 percent, or more than 88 percent, or more than 90 percent with SEQ ID No.1 and has the same function with SEQ ID No. 1.
4. A method for modifying the stem node character of a plant and/or modifying the plant type of the plant and/or increasing the yield or biomass of the plant, which comprises introducing the PtCYP714F1 gene sequence of claim 1 into a plant cell by transgenic technology; alternatively, the method comprises increasing expression of a PtCYP714F1 gene sequence in the plant.
5. A method according to claim 4, wherein the method comprises increasing the number of stem nodes, and/or increasing the number of internodes, and/or increasing the number of axillary buds or leaves of the transgenic plant.
6. A method for constructing a transgenic plant, comprising introducing the PtCYP714F1 gene sequence of claim 1 into a plant cell by transgenic technology.
7. A method for constructing transgenic rice, comprising the steps of:

(1) b L AST comparison is utilized to find a gene sequence which is homologous with the rice OsCYP714D1 from a sequencing poplar variety, a primer is designed, and a PtCYP714F1 gene is obtained by amplification from the sequencing poplar variety, and sequencing verification is carried out (SEQ ID No. 1);

(2) cloning a promoter proD1 of an OsCYP714D1 gene from rice, constructing a plant expression vector, transferring the PtCYP714F1 gene into rice callus, and obtaining a plurality of resistant strains by inducing callus differentiation and multi-round hygromycin screening;

(3) performing PCR identification on the screened resistant strains, and determining the PCR positive strains as PtCYP714F1 transgenic strains;

(4) carrying out RT-PCR detection on the expression level of the PtCYP714F1 gene in the transgenic line, and determining the expression quantity of the PtCYP714F1 gene in different transgenic lines;

(5) phenotypic analysis and comparison were performed on transgenic lines and controls (eui mutants and wild type) and internode length and number of stem nodes were counted.
8. A plant cell obtained by the method of claim 6 or 7.
9. A vector comprising the PtCYP714F1 gene sequence of any one of claims 1 to 3.
10. A plant cell comprising the PtCYP714F1 gene sequence of any one of claims 1 to 3.