CN115011610B

CN115011610B - Application of MdTCP17 and MdWOX11 in interaction regulation and control of MdLBD29 gene expression and adventitious root generation

Info

Publication number: CN115011610B
Application number: CN202210689983.9A
Authority: CN
Inventors: 张东; 毛江萍; 李民吉; 马娟娟; 邢利博; 安娜; 王超; 艾思源; 孟子奇; 史继东
Original assignee: Northwest A&F University
Current assignee: Qianxian Qiangsheng Fruit Industry Co ltd
Priority date: 2022-06-17
Filing date: 2022-06-17
Publication date: 2023-06-23
Anticipated expiration: 2042-06-17
Also published as: CN115011610A

Abstract

The invention belongs to the field of biotechnology, and discloses an application of MdTCP17 and MdWOX11 in interaction regulation and control of MdLBD29 gene expression and adventitious root generation, wherein the MdWOX11 and MdWOX 17 interact, and MdWOX11 induces MdLBD29 expression; the apple MdLBD29 promoter has a nucleotide sequence of SEQ ID NO.1, and is derived from apple stock Malus toringoides, and MdTCP17 inhibits the transcriptional activation of MdLBD29 by MdWOX 11. The invention firstly researches whether the apple MdWOX11 interacts with the MdTCP17 protein or not and whether the MdTCP17 regulates and controls the transcription regulation of the MdWOX11 or not, and has important significance for solving the development regulation mechanism of the adventitious roots of apple stocks. Meanwhile, the invention has potential value for developing a clone stock breeding regulation technology and molecular breeding of stocks with excellent adventitious root generating capacity.

Description

Application of MdTCP17 and MdWOX11 in interaction regulation and control of MdLBD29 gene expression and adventitious root generation

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an application of MdTCP17 and MdWOX11 in interaction regulation and control of MdLBD29 gene expression and adventitious root generation.

Background

Currently, apples (Malus domestica Mill) are one of fruit trees with commercial importance in the world and are also main fruit crops in temperate regions of the world, and statistics of world grain and agriculture organization (FAO) show that apples in China are rapidly produced and developed, the area of apples in the world in 2017 is 493.38 ten thousand hectares, the yield is 8313.93 ten thousand tons, the total area of apples in 2017 in China is 222.02 ten thousand hectares, the total yield is 4139.00 ten thousand tons, the total area and the total yield in the world are 45.00 percent and 49.78 percent respectively, and the first world is ranked (FAOSTAT data). The natural ecological environment of China is favorable for the growth and development of apple trees, and the popularization of excellent stocks is of great importance, so that the induction of adventitious roots is a key place for stock breeding. The research on the molecular mechanism of adventitious roots is not only beneficial to solving the problem of difficult rooting of certain apple stocks, but also can discuss the regulation and control network of adventitious roots through the molecular biology technology, thereby providing a favorable measure for promoting root system development in production.

Expression of WOX family genes all have spatiotemporal specificity and may all be involved in regulation of plant apical development by different mechanisms of action. The regulatory network between WOX family genes and related genes is not well understood. The TCP family genes are a class of transcription factors encoding plant specificities, and family members contain a bhlh motif that can bind DNA or promote interactions between proteins. At present, the research on the functions of TCP genes is more and more increasing, and the TCP genes regulate the growth and development of plants, have important influence on the growth of meristematic tissues and collateral organs and are beneficial to generating new agricultural characters. TCP proteins can act as transcriptional activators or transcriptional repressors, but regulatory networks between TCP family genes and related genes remain to be resolved.

LBD is a class of transcription factors specific for higher plants that is able to respond to a number of signals such as hormones, nutritional elements, biotic and abiotic stresses, etc., LBD (Lateral Organ Boundaries Domain) is a class of transcription factors specific for higher plants, and members of this family typically contain a conserved LOB domain at the N-terminus. Plant LBD family members can be classified into Class I and Class II 2 major classes based on sequence similarity. The existing research results show that the class I LBD members are mainly expressed on the primordial boundary of the collateral organs, and regulate the initiation of collateral organs including collateral root lateral buds and the like, so that the growth and development of a plurality of organs of plants are regulated. Class II LBD members are involved in plant anthocyanin synthesis and nitrogen metabolism regulation. Many studies have shown that LBD family members are involved in a variety of biological processes, playing an important role in hormone-mediated plant secondary metabolism, plant collateral organ development, plant nutrient uptake, and stress processes. The LBD family is very huge, the functional difference among all members is larger, but most genes involved in root development are concentrated in the aspect of lateral root development regulation, and the report on the regulation effect of adventitious roots is little.

Through the above analysis, the problems and defects existing in the prior art are as follows: there are few reports of WOX11 interacting proteins in the prior art, no report of TCP17, and few reports of regulation of adventitious root occurrence by LBD family. Aiming at the industrial problem of difficult rooting of apple stocks, the prior art focuses on optimizing a culture medium formula and applying reagents to induce rooting, and related researches on molecular levels are not yet carried out, the invention finds three key transcription factors for regulating root system development from the gene level, analyzes the regulation relation among the three genes, preliminarily answers the scientific problem of the molecular mechanism of adventitious roots of the apple stocks, applies the molecular mechanism to the development of the adventitious root regulation technology, and has important guiding significance for solving the industrial problem of difficult rooting of the apple stocks.

Disclosure of Invention

Aiming at the problems existing in the prior art, the invention provides an application of MdTCP17 and MdWOX11 in interaction regulation and control of MdLBD29 gene expression and adventitious root generation.

The invention is realized by the application that the MdTCCP 17 and MdWOX11 interact to regulate the MdLBD29 gene expression and the generation of adventitious roots, wherein the MdTCCP 17 interacts with the MdWOX11 protein.

Further, the MdWOX11 induces expression of MdLBD 29.

Further, the MdTCP17 inhibits transcriptional activation of MdWOX11 on MdLBD 29.

Further, the overexpression of MdWOX11 and interference with MdTCP17 promotes expression of MdLBD 29.

It is another object of the present invention to provide an apple MdLBD29 promoter, wherein the nucleotide sequence of the apple MdLBD29 promoter is SEQ ID No.1.

Further, the apple MdLBD29 promoter nucleotide sequence is from apple stock malus haliotidis.

It is another object of the present invention to provide a method of validating that MdWOX11 is overexpressed and that MdTCP17 is interfered with the expression of MdLBD29, the method of validating that MdWOX11 is overexpressed and that MdTCP17 is interfered with the expression of MdLBD29 comprising:

apple tissue culture Miao Shunshi transformation was used to confirm overexpression of MdWOX11 and to interfere with MdTCP17 to promote expression of MdLBD 29; wherein, the apple instant transformation material is apple tissue culture seedling.

Further, in the bacterial liquid of the apple tissue culture seedling instantaneous conversion system, the apple tissue culture seedling is infected by adopting a vacuum infiltration mode; preferably, the concentration of the bacterial liquid OD600 of the MdTCC 17 interference carrier is 0.6.

Further, preferably, the infection time is 8 to 10 minutes;

further, preferably, the post-infestation apple leaf cultivation temperature is 21 ℃.

In combination with the above technical solution and the technical problems to be solved, please analyze the following aspects to provide the following advantages and positive effects:

first, aiming at the technical problems in the prior art and the difficulty in solving the problems, the technical problems solved by the technical proposal of the invention are analyzed in detail and deeply by tightly combining the technical proposal to be protected, the results and data in the research and development process, and the like, and some technical effects brought after the problems are solved have creative technical effects. The specific description is as follows:

the invention adopts apple transcription factor MdWOX 17 to interact with transcription factor MdWOX11 for the first time, and jointly verifies that MdWOX 17 and MdWOX11 protein interact through yeast double hybrid and double molecular fluorescence complementation and Co-IP. The dual luciferase assay demonstrated that MdWOX11 induced expression of MdLBD29, while MdTCP17 inhibited transcriptional activation of MdLBD29 by MdWOX 11. Apple transient transformation demonstrated overexpression of MdWOX11 and interfered with MdTCP17 to promote expression of MdLBD 29.

The invention firstly researches whether the apple MdWOX11 interacts with the MdTCP17 protein or not and whether the MdTCP17 regulates and controls the transcription regulation of the MdWOX11 or not, and has important significance for solving the development regulation mechanism of the adventitious roots of apple stocks.

Secondly, the technical scheme is regarded as a whole or from the perspective of products, and the technical scheme to be protected has the following technical effects and advantages:

the invention has very important research significance for discussing the interaction of MdTCP17 and MdWOX11 and regulating the expression of MdLBD29 genes and the generation of adventitious roots. The invention has potential value for developing a clone stock breeding regulation technology and molecular breeding of a stock with excellent adventitious root generating capacity.

Thirdly, as inventive supplementary evidence of the claims of the present invention, the following important aspects are also presented:

(1) The technical scheme of the invention fills the technical blank in the domestic and foreign industries:

the invention discovers that the MdWOX11 and MdTCCP 17 proteins interact for the first time, and proves that MdTCCP 17 inhibits the transcriptional activation of MdWOX11 on MdLBD 29. The invention fills up the technical blank of domestic and foreign industries from the gene regulation level, and reveals the industrial problem of difficult rooting of apple rootstocks, and has creative research significance.

(2) The technical scheme of the invention solves the technical problems that people are always desirous of solving but are not successful all the time:

the apple rootstock rooting difficulty is an apple industry problem which is always desired to be solved, and the invention preliminarily answers the problem through a gene level regulation and control way. Namely, in apple stocks which are difficult to root, mdWOX11 and MdTCCP 17 interact, and MdTCCP 17 inhibits the transcriptional activation of MdWOX11 on MdLBD29, thereby inhibiting the rooting of the apple stocks. By deeply analyzing the physiological molecular mechanism and regulation rule of the adventitious roots of the rootstock, developing and optimizing the efficient regulation technology of the adventitious roots, researching the integrated matching technology, widely applying the adventitious roots technology to the sugar-free tissue culture rooting and layering propagation of the apple rootstock, realizing the mass production of the apple rootstock material with difficult rooting and forming a mature seedling propagation system.

(3) The technical scheme of the invention overcomes the technical bias:

the prior art focuses on methods such as hormone and chemical reagent treatment on apple stocks to promote apple rooting, and the problem of difficult rooting of apple stocks is not discussed from the aspect of genes. The invention overcomes the industrial problem of difficult rooting of the apple rootstock in the traditional rooting method, and analyzes the apple rootstock from the gene level for the first time, thereby providing an internal mechanism of the apple rootstock difficult to root in the gene regulation level. Namely, in apple stocks which are difficult to root, mdWOX11 and MdTCCP 17 interact, and MdTCCP 17 inhibits the transcriptional activation of MdWOX11 on MdLBD29, thereby inhibiting the rooting of the apple stocks.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments of the present invention will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph of the interaction between the yeast double hetero-verification protein MdWOX11 and MdTCP17 provided by the embodiment of the invention;

FIG. 2 is a cross-plot of the two-molecule fluorescent complementary verification protein MdWOX11 and MdTCP17 provided by the embodiment of the invention;

FIG. 3 is a graph showing interactions between Co-IP verification proteins MdWOX11 and MdTCP17 provided by an embodiment of the invention;

FIG. 4 is a graph showing that the co-expression of MdWOX11 and MdTCCP 17 inhibits MdLBD29 by using double luciferases provided by the embodiment of the invention;

FIG. 5 is a graph of apple transient transformation verification over-expression of MdWOX11 and interference of MdTCCP 17 to promote MdLBD29 provided by the example of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

In view of the problems existing in the prior art, the invention provides an application of MdTCP17 and MdWOX11 to mutually regulate and control MdLBD29 gene expression and adventitious root generation, and the invention is described in detail below with reference to the accompanying drawings.

1. The embodiments are explained. In order to fully understand how the invention may be embodied by those skilled in the art, this section is an illustrative embodiment in which the claims are presented for purposes of illustration.

The application of the interaction of the apple MdTCCP 17 and the MdWOX11 in the embodiment of the invention for regulating the MdLBD29 gene expression and the generation of adventitious roots proves that the interaction of the MdWOX11 protein and the MdCP 17 is proved by double-hybrid yeast, double-molecule fluorescence complementation and co-immunoprecipitation tests, and the negative control can not be combined (see figures 1,2 and 3).

In the embodiment of the invention, a 2652bp MdLBD29 promoter nucleotide sequence is obtained by cloning in Malus pumila stock Malus pumila, and the nucleotide sequence contains a binding element WOX-box of MdWOX 11. It was found by double luciferase assay that MdWOX11 can bind to the promoter of MdLBD29 and induce expression of MdLBD29 (see fig. 4).

In the invention, in a dual-luciferase system, mdTCP17 and MdWOX11 are added simultaneously, and the co-expression of MdTCP17 and MdWOX11 is found to inhibit the induction of MdWOX11 on MdLBD29, so that the MdTCP17 is presumed to inhibit the transcriptional activation of MdWOX11 on MdLBD29 (see figure 4).

Apple tissue culture Miao Shunshi transformation provided by the embodiment of the invention proves that the MdWOX11 is over-expressed and the MdTCCP 17 is interfered to promote the expression of MdLBD 29. The method is realized specifically as follows: and (3) infecting the apple tissue culture seedlings by adopting a mode of vacuum infiltration by using bacterial liquid mixed with heavy suspension, wherein the OD600 concentration of the infecting bacterial liquid is 0.6, the infecting time is 8-10 min, the bacterial liquid is sucked by sterile filter paper after the infection is finished, the tissue culture seedlings are transferred to an expanding propagation culture medium, and the tissue culture seedlings are placed in a biochemical incubator at 21 ℃ for dark culture for 3d. The whole tissue culture seedling was subjected to RNA extraction and reverse transcription, and the expression level of MdLBD29 was analyzed. The results showed that MdWOX11 was overexpressed and that interference with MdTCP17 promoted expression of MdLBD29 (see fig. 5).

The nucleotide sequence of the SEQ ID NO.1MdLBD29 promoter provided by the embodiment of the invention is as follows:

TATCCTATGATAGTTTAGGTATTATGTAACCTTCATAGATGATTGTACCCGATACACATGGATTTTTCCATTAAGAAATAAATCAGAAGTTTTTGCAATCTTTGTTCAGTTTCATGCCTATTTGTGTACCCAGTTTTCTGCTATAGTCAAAATATTCCAAAGTGATGGTGGTGGTGAATGTACAAGCAATAAGTTTCAACAATATTTGTCAAAACATGGTATTTTACATCACAAATCATGTCCATATACACCTGAACATAATGGTTTGGCAGAAAGGAAACATAGGCATCTTGTAGAAACAGCAATTACACTTCTTCAAACTGCAAAATTGCCTAACCAATTTTGGTTTCATGCCTGTGCAACTGCCTCCTATCTGATCAATAGGAAACCTAGTGCTTCTTTAAATATGCAGTCACCATTTCAAAAGTTATATAACACAGTTCCTGAGATCAAACATCTTAGGGTCTTTGGTTGTGCATGTTTTCCCCTTTTAAAACCCTATAACATTTCTAAACTTCAGCCTAAAACAACAACTTGTGTGTTTTTGGGATATGTAGGCCAACATAAGGGTTTCATATGTCTAGATATTGCTCATGGTAAAATTTATGTGGCTAGGCATGTTTTGTTTGATGAATCTACTTTTCCATATTCAAACCCTAGTTTACTTCAACGCTTAAAATTGTCTTCTGCATCATCACAGTTACATTCACCAAATACTTCAAGTTACCCCCGTGTCACTTTCGTGTAAATCTATTAAGGACCTATTATTAATGGGTTCTTCTCGTGTGACCTGATAAAGATCTGATTAGTTATTATGTTGACCCAAAACCCTTTATTTTTGTATCGTTTACGTGTTGTGTAAGAAATTGCCAGGTTTATATGGATCCACCCCTATATGTGTGTGTGTATATATTTATAATATTAATTTACTACCGGTCATCCATTCATTCATGTGCTTAATTAAGTGCTTTGGTGGTAAGAGCTTGTCCAGTAAGCTGATCTGCATGCAGAAGCAAAAGCAGAAAGTTGAACTCAGAATTATCCGTGACATGGGAATGTAGCTTGTGTTGCAAACTGTCATTTTCCTTTGTTAGGATGACGACCTTCTCATTCATTTGAGTTGAAACCTCTTCCCTTGTGGCTTCTAATCTATGCACCAAGTCACAGTACAAAATTTTTATATATGTGCCCTAAGGAGGTGGCAGCTGGTCAATAATAACTCTGATCCCACAGGAGCACCTGTTGTATTATTCATCCATGCATTCATTCATTGAAGTTTACCTTGGATAAATTGGGTCATATACTTGATTGACCACGTTCTATTTTGGTTTTGGTAAAACTGTCTTGTGAGGGACTAGCATAGTTGAAGTGAGGACTTTAGGGTTTTCACCCCATACTATGGGCGCGTTGAGAAAATCTCATATTTTTACTTGTTTGATTTTGTTTATTGATACGAGTGATTGTCTAGTGTTAAAATTACCATACCAAAAGCTTGAACTTTAGAGTCCAGCTAGTCTCATAATACGTAATTGCTTAGATATAAAATTTTAAAGCACCAAACTCTGAAACTTTAAACCTAAAATCGAAGCTCTAAAGGATTATTCTTGATAAGTCAATCATTTGAAAAAAAACGGACCTCCTATAAAAAAAAAAAAAAAATCTCTATGAGATGTTACTTGTTAGTTGATGCTAACTGAACTAGTTTTTTTTCCCTAATTAACAAAGTATCTGGACTGCAGCAACTAGGATCAGTCAACAAATCCAAAATTTTGCAGTTCTTGTGGTTCAAGTTGAGGCCAGTAGTGACTGAAGGATTTAATTTGTGACAGGTGAGATTTTGGTTTGTGAAAACGATGGCCTTATTAAACCCACTTTTGACTAGTTTTCCTTTTTGTCTCATGTGATCAATGGCATGTTGTATAACGCACGAGGATAACACAACTGCACTCACTTCATTATAGACTTCAAGATCAATCCTTCAGCTACAAGAAAGCACTTATCTCGTCATCTTCTTACGACCCAGTTTCCAAAATCTCGACCTAATCAGAAAATCTTACCTATCTCCGTTTATATTTTTGTGCCCATCTCACTAATTATGTCCTCAATCACATAACCATGAGATAAAAACATACGTATGTAGTATATTCTTCACAAGTAAGTCTTCCTCGTAACAAACACCAAACCAAACCAAAGCAGCACCAATCTTCTTCACCAAGTTGGAGGATTGGAGTCTTGAGCTTGAACAACACTAATGACTAATTAGTTTAATAAATCACTTGCGTATATATGTTTGCAACATTATAAACCCTATTTTAAAACCATATTGCTCAAGGTAACTGAATCATTAGTAATTGTCGGTAGAAAAATATGCCAGAAGAGCTGTGGCAAGTGCAGTGAGTGAACCACAATGTCACAAGGCGACAATTTGCAGCATTTCCCAGGTGCGGCGTTCGGCGGAAAATATAAACATATTCAAACCCTAAAATCAAACTTTGCAATTAAAAAATTGCTAATCATACTCGGTAGCCATTAATCATCCACGCTGGAAAAATATAAAACGAAAAAAGAAATGATTGTATTAAAAATATGATGAATGCTTAAAAAAAGTAGACCCCATGATATTACATAAACCATTAAAAATTGTCCCCTTAG

the technical scheme of the invention is further described below with reference to specific embodiments.

Example 1

The embodiment of the invention provides an application of apple MdTCCP 17 and MdWOX11 in interaction regulation and control of MdLBD29 gene expression and adventitious root generation. Yeast double hetero examined whether there was protein interaction between MdTCP17 and MdWOX 11. pGAD424-MdWOX11 and pGBT 9-MdTCCP 17 vectors were constructed first. The coding region sequences of MdWOX11 and MdTCCP 17 were ligated into pGAD424 and pGBT9 vectors, respectively, by double digestion. pGAD424-MdWOX11 plasmid and pGBT 9-MdTCCP 17 plasmid were extracted separately and co-transformed into Y2H yeast (Clontech, paloAlto, CA, USA). Because pGBD-MdWOX11 has a self-activating effect, after the self-activating area of MdWOX11 is removed, mdWOX11 is divided into an N end and a C end (marked as MdWOX11N and MdWOX 11C), and 5 combinations are MdWOX11N+MdTCP17, mdWOX11 C+MdWOCP 17, mdWOX11 N+pGBJ9 empty vector, pGAD424 empty vector+MdWOCP 17, pGBT9 empty vector+pGAD 424 empty vector respectively. The 5 combined transformed yeasts were cultured in SD/-Leu/-Trp medium and transferred to SD/-Leu/-Trp/-His/-Ade yeast medium, after which the yeast spots grown on this medium were cultured at 28℃for 8-12 h, after which 2. Mu.L were pipetted onto DDO/X, DDO/X/A and QDO/X-a-Gal/AbA yeast medium, respectively, and the interaction of the protein MdWOX11 with MdTCC 17 was determined from the growth of the yeast spots. As shown in FIG. 1, the results showed that the MdTCP17 and MdWOX11 proteins can interact, pGBD-MdWOX11 was cultured on a 4-deficient medium (SD/-Leu/-Trp/-His/-Ade) and the yeast plaques developed a blue color (see FIG. 1). The N-terminus of MdWOX11 interacted with MdTCCP 17, and yeast cultures transferred into pGAD-MdTCCP 17 and pGBD-MdWOX11N showed plaques on QDO/X/A screening medium, and the yeast system demonstrated interaction of MdWOX11 protein with MdTCCP 17 protein (see FIG. 1).

Example 2

Bimolecular fluorescence complementation (BiFC) further explored whether there was interaction between the proteins MdWOX11 and MdTCP17, the bimolecular fluorescence complementation assay inserted the full length coding sequences of the MdWOX11 and MdWOX 17 genes into vector 35S: pSPYNE-nYFP and 35S: in pSPYCE-cYFP, the constructed vector and empty vector are transformed into an Agrobacterium strain EHA105, 500 mu L of overnight-cultured Agrobacterium solution is taken to 50mL of culture medium, the culture medium is cultured until the OD600 = 0.6-0.7, the culture medium is centrifuged at 5000rpm at room temperature for 10min, the suspension is washed and centrifuged, the suspension containing different vectors is regulated to be about OD600 to 0.3-0.5 by the suspension, the three combinations are respectively mixed by MdTCP17-nYFP+MdWOX11-cYFP, mdCTP 17-nYFP+pSPYCE-cYFP empty vector and pSPYNE-nYFP empty vector +MdWOX11-cYFP, and the corresponding suspensions are respectively mixed in equal volumes and then are stood for 1-2 h. The 3 different combinations of agrobacteria were mixed and injected into the leaf of Nicotiana benthamiana, then incubated at 23℃in the dark for 2-3 days, and YFP fluorescence of the leaf of Nicotiana benthamiana at the injection site was detected under a 488 nm-excited confocal laser scanning microscope (Zeiss LSM 510Meta, jena, germany). The bimolecular fluorescence complementation results are shown in FIG. 2, and very strong yellow fluorescence signals are found in the nuclei of tobacco leaves co-transformed with MdWOX11-nYFP and MdTCP17-cYFP, and the nuclei were stained with the basic dye DAPI of the nuclei, and the nuclei exhibited blue signals, and the picture of the integration of the two fields of GFP and DAPI revealed that the interaction signals were indeed present in the nuclei. No fluorescent signal was seen in Nicotiana benthamiana cells transformed with either the empty vector nYFP+MdTC17-cYFP or the MdWOX11-nYFP+ empty vector cYFP, and the BiFC assay demonstrated that the MdWOX11 protein interacted with the MdTC17 protein in vivo (see FIG. 2).

Example 3

To further clarify the relationship between MdWOX11 and MdTCP17, a Co-immunoprecipitation (Co-IP) experiment was performed. Firstly, full-length coding sequences of MdWOX11 and MdWOX 17 genes are respectively inserted into vectors pCXSN-Myc and pCAMBIA2300-GFP, after separation of arabidopsis protoplasts, plasmids expressing MdWOX 17-GFP and MdWOX11-Myc proteins are transferred into the arabidopsis protoplasts through a PEG mediated method, and cells are collected for Co-IP test after 18h of culture. Finally, detection is carried out through western-blot. As a result, as shown in FIG. 3, the MdWOX11 protein fused to Myc tag protein was immunoprecipitated by the MdTCP17 protein fused to GFP tag protein, whereas no hybridization band was detected in combination with cotransverse from pCXSN-Myc and pCAMBIA-TCP17-GFP plasmids (see FIG. 3).

Example 4

Given that MdWOX11 transcriptional regulates MdLBD29, then MdWOX11-MdTCP17 interaction is followed by MdTCP17 to determine whether MdWOX11 has an effect on the transcriptional activity of MdLBD 29. Subsequently, a dual luciferase reporter assay was performed in Nicotiana benthamiana leaves to verify this assumption. First, the MdLBD29 promoter sequence is inserted into pGreen II0800-LBD29-LUC vector, and the MdLBD29 promoter sequence is shown as SEQ ID NO.1. Full-length coding sequences of the MdWOX11 and MdTCCP 17 genes are inserted into vectors pGreen II62-SK, and pGreen II0800-LBD29-LUC, pGreen II62-SK, pGreen II62-WOX11-SK and pGreen II 62-TCP17-SK are respectively transferred into agrobacterium GV 3101. The treated bacterial solutions were divided into 4 groups, respectively, pGreen II0800-LBD29-LUC+pGreen II62-SK, pGreen II0800-LBD29-LUC+pGreen II62-WOX11-SK, pGreen II0800-LBD29-LUC+pGreen II 62-TCP17-SK, pGreen II0800-LBD29-LUC+pGreen II62-WOX 11-SK+pGreen II 62-TCP17-SK, and the LUC/REN values were measured for tobacco leaves after 3d infection, as shown in FIG. 4, the LUC/REN levels were significantly lower than those of MdWOX11-SK and ProLBD29-LUC after co-conversion of MdWOX11-SK, mdWOCP 17-SK, and ProLBD29-LUC, indicating that MdWOX11 inhibited transcription activation activity on MdLBD29 (see FIG. 4).

Example 5

To further confirm that MdWOX11 and MdTCP17 interaction followed by MdTCP17 affects the transcriptional activity of MdWOX11 on MdLBD29, apple tissue culture Miao Shunshi transformation experiments were performed. Firstly, the constructed MdWOX11 over-expression and MdTCP17 interference vector are respectively transformed into agrobacterium competent EHA 105. mu.L of an overnight culture agrobacteria solution was taken and cultured in 50mL of a medium until OD600 = 0.8 or so, centrifuged at 5000rpm at room temperature for 10min, rinsed with the suspension, centrifuged, and OD600 to 0.6 or so was adjusted with the suspension. And (3) infecting the apple tissue culture seedlings in a bacterial liquid of the apple tissue culture seedling instantaneous conversion system in a vacuum infiltration mode. The specific operation is that the apple tissue culture Miao Cong is taken out of a tissue culture bottle, is put into a needle tube containing agrobacterium tumefaciens heavy suspension for repeated whipping, is infected by a vacuum infiltration mode for 8-10 min, is dried by a sterile filter paper after infection, is put into an expanding culture medium for dark culture for 3d, the culture temperature during the process is 21 ℃, and the formula of the expanding culture medium is 4.43g MS powder, 30g sucrose, 7.8g agar, 0.2mg 6-BA and 0.2mg IBA per liter. And taking out the whole apple tissue culture seedling from the propagation culture medium, grinding the whole apple tissue culture seedling into powder by using liquid nitrogen, extracting RNA and reversely transcribing the RNA into cDNA. Quantitative primers were designed and quantitative PCR was performed. As a result, as shown in FIG. 5, over-expressing MdWOX11 promoted the expression of MdLBD29, over-expressing MdWOX11 and interfering MdTCCP 17 inhibited the expression of MdLBD29 (see FIG. 5).

2. Application example. In order to prove the inventive and technical value of the technical solution of the present invention, this section is an application example on specific products or related technologies of the claim technical solution.

It was found at the gene level that the expression of MdWOX11 and MdLBD29 correlated positively with adventitious root formation ability, and MdTCP17 correlated negatively with adventitious root formation ability. In apple stocks which are difficult to root, the expression quantity of the MdTCCP 17 is high, the MdTCCP 17 interacts with MdWOX11 protein, and the MdTCCP 17 inhibits the transcriptional activation of MdWOX11 on MdLBD29, so that the occurrence of adventitious roots of the apple stocks is inhibited. The molecular mechanism of adventitious root generation has been successfully applied in apple industry, we promote the generation of adventitious root of apple stock by promoting the expression of two genes of MdWOX11 and MdLBD29 through hormone treatment and inhibiting the expression of MdTCC 17 in production practice, and successfully develop and optimize a set of adventitious root regulation and control technical system.

3. Evidence of the effect of the examples. The embodiment of the invention has a great advantage in the research and development or use process, and has the following description in combination with data, charts and the like of the test process.

The exogenous auxin treatment can promote the expression of MdWOX11 and MdLBD29, inhibit the expression of MdTCP17, obviously promote the occurrence of adventitious roots, and mainly promote the occurrence of adventitious roots by promoting the number of adventitious roots and the occurrence rate of adventitious roots. And secondly, the expression of MdTCCP 17 can be promoted by exogenous cytokinin treatment, the expression of MdWOX11 and MdLBD29 can be inhibited, and the occurrence of adventitious roots can be obviously inhibited. In some apple stocks which are difficult to root, such as SH-series stocks, the number of adventitious roots of apples can be obviously increased by treating exogenous auxin and cytokinin inhibitors, and the adventitious roots are promoted mainly by promoting the expression of MdWOX11 and MdLBD29 and inhibiting the expression of MdTCC 17.

The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.

<110> university of agriculture and forestry science and technology in northwest

<120> use of MdCP17 and MdWOX11 to control MdLBD29 Gene expression and adventitious root Generation by interaction

<160>1

<210>1

<211>2652

<212>DNA

<213> Artificial sequence (Artificial Sequence)

<400>1

Claims

1. MdTCP17 protein and MdWOX11 protein interact in negative regulationMdLBD29The use of gene expression and adventitious root generation, characterized in thatMdTCP17Protein inhibitionMdWOX11Protein pairMdLBD29Transcriptional activation of genes, saidMdTCP17Protein negative regulation of apple adventitious root.

2. The method of claim 1, wherein the MdTCP17 protein and MdWOX11 protein interact in negative regulationMdLBD29The use of gene expression and adventitious root generation, characterized in that the MdWOX11 protein inducesMdLBD29Expression of the genes.

3. The method of claim 1, wherein the MdTCP17 protein and MdWOX11 protein interact in negative regulationMdLBD29The use of gene expression and adventitious root generation, characterized in thatMdLBD29The nucleotide sequence of the promoter of the gene is shown as SEQ ID NO.1.

4. The method of claim 1, wherein the MdTCP17 protein and MdWOX11 protein interact in negatively regulated applesMdLBD29Gene expression and adventitious root genesisIs characterized in that the overexpression is achieved byMdWOX11Gene and interferenceMdTCP17Gene promotionMdLBD29Expression of the genes.

5. A method of promoting adventitious roots of an apple, the method comprising:

transformation using apple tissue culture Miao ShunshiMdWOX11Gene overexpression vectorMdTCP17The gene interferes with the vector.

6. The method for promoting adventitious roots of apples to occur according to claim 5, wherein the apple tissue culture seedlings are infected by vacuum infiltration in a bacterial liquid of an apple tissue culture seedling transient transformation system;

the saidMdTCP17The concentration of the agrobacteria liquid OD600 of the gene interference vector is 0.6.

7. The method for promoting adventitious roots of apples to occur according to claim 6, wherein the infection time is 8-10 min.

8. The method for promoting adventitious roots of apples according to claim 6, wherein the temperature for cultivating the tissue culture seedlings of apples after infection is 21 ℃.