CN104004076B - A kind of apocarya MADS-box class transcription factor CiAG and encoding gene thereof and application - Google Patents

Abstract

The invention discloses a kind of apocarya MADS box class transcription factor CiAG and encoding gene thereof and application.This apocarya MADS box class transcription factor CiAG, is to have the protein of aminoacid sequence described in the SEQ ID NO.2 in sequence table, and its encoding gene is the DNA sequence of CiAG genes of SEQ ID NO.1.Apocarya MADS box class transcription factor CiAG gene can be used for cultivating early blossoming, dwarf plant kind.CiAG is specific expression gene, and its expression pattern is relevant to the stage of development of tissue and plant.The transgenic arabidopsis of overexpression CiAG is compared with matched group, bloom more Zao than WT lines, bloom when four basal leaves occur, and plant shows the characteristic of dwarfing, illustrate that CiAG can control the flowering transition of plant, affect the florescence of plant and nourish and grow, and then the reproductive growth of regulation and control plant.

Description

A kind of apocarya MADS-box class transcription factor CiAG and encoding gene thereof with Application

Technical field

The present invention relates to plant genetic engineering field, be specifically related to a kind of apocarya MADS-box class transcription factor CiAG and encoding gene thereof and application.

Background technology

Apocarya child's phase is the longest, needs about 15 years, and male flower is grown early, and female flower development is late.It addition, shell mountain core Fructus Persicae is the different flowering plant of hermaphroditism, there are female antetype, male antetype and three kinds of different types of male and female homotype, and male flower is main Raw on the staminate inflorescence of raw branch middle and lower part the previous year, and female flower once raceme, raw on the top of fruitful branch.Thus may be used Seeing, apocarya female flower and male Floral development have the feature of uniqueness, study its flower development gene, for cultivating early blossoming Early real kind and having great importance shortening the juvenile phase.

MADS-box is the DNA binding structural domain that a conservative is the strongest, as far back as yeast Mini Chromosome Maintenance1 (MCM1), arabidopsis AGAMOUS (AG), Antirrhinum majus L. DEFICIENS (DEF) and serum human response factors Finding in Serum ResponseFactor (SRF), the functional gene therefore with this structure is named as MADS-box gene. MADS-box has important effect in fruit tree shortening the juvenile phase, but whether this gene can play a role in dwarfing fruit trees There is not been reported.

Summary of the invention

Goal of the invention:

It is an object of the invention to provide a kind of apocarya MADS-box class transcription factor CiAG.

It is a further object of the present invention to provide the encoding gene of this transcription factor CiAG.

It is yet another object of the invention to provide the application of this transcription factor.

The purpose of the present invention can be achieved through the following technical solutions:

A kind of apocarya MADS-box class transcription factor provided by the present invention, named CiAG, derive from shell Semen Caryae Cathayensis (Carya illinoensis (Wangench.) K.Koch) improved seeds ' Ma Han ', aminoacid sequence such as SEQ ID Shown in NO.2.

The encoding gene of apocarya MADS-box class transcription factor of the present invention, its cDNA sequence such as SEQ Shown in ID NO.1；Sequence contains the maximum open reading frame of 684bp, encodes 227 aminoacid shown in SEQ ID NO.2 Residue sequence.

Expression vector containing CiAG genes of SEQ ID NO.1 of the present invention.

Described expression vector is preferably by the encoding gene of described apocarya MADS-box class transcription factor CiAG It is inserted into gained between Kpn I and Sac I restriction enzyme site of pCAMBIA1301 carrier.

Host Strains refers to the Agrobacterium tumefaciems EHA105. proceeded to by pCAMBIA1301-CiAG

The primer of amplification CiAG cDNA total length is:

VvAG-ORF sense:5'-ATGGGGAGGGGGAGGATAGAAA-3'

VvAG-ORF antisense:5'-CGCCATAACAGGGCAATAACCT-3'

The qPCR primer of the CiAG related in real-time fluorescence quantitative RT-PCR analysis is:

VvAG qPCR sense:5'-AGGCTGCTACTCTACGACAAC-3',

VvAG Qpcr antisense:5'-GGTTCCTCTCATTCTCCGCTATC-3'.

Above-mentioned apocarya MADS-box class transcription factor CiAG, its encoding gene, expression containing encoding gene carry Body application in cultivating early blossoming, dwarf plant.

Beneficial effect:

The present invention is cloned into a MADS-box class transcription factor CiAG gene in apocarya, and CiAG may participate in Most of process of the reproductive developments such as apocarya flower development, fruit development.CiAG encoding gene is at male flower, female flower and young fruit In specific expressed.Overexpression CiAG encoding gene in transgenic arabidopsis, compared with matched group, florescence significantly shifts to an earlier date, Plant is downgraded.The result shows that CiAG gene can control the one-tenth China transition process of plant, plant can be made to downgrade simultaneously.

The CiAG of the present invention has for the apocarya cultivating early blossoming, dwarf plant kind particularly early blossoming precocity is downgraded Significant, it is with a wide range of applications in terms of crop breeding.

Utilize the plant expression vector of the present invention, by CiAG gene transfered plant body, it is possible to obtain bloom in advance, plant The transfer-gen plant downgraded.

Accompanying drawing explanation

Fig. 1 CiAG gene is at the expression characterization of apocarya

1, leaf；2, current-year branch；3, female flower；4, male flower；5, young fruit

The structure of Fig. 2 .CiAG overexpression expression vector

Fig. 3. turn CiAG gene arabidopsis and matched group RT-PCR detection.

A:CiAG special primer RT-PCR product；B:18S product；WT: wild type；1-5: transgenic

Fig. 4 turns CiAG gene arabidopsis and the Phenotypic Observation of comparison

WT: wild type；T: transgenic line

Detailed description of the invention

In following Examples, all of method is without special instruction, is conventional method

The clone of embodiment 1 apocarya CiAG gene and qualification

Experiment material is apocarya kind ' Ma Han ', has cloned the conservative fragments obtained (not according to this laboratory Positive sea etc., 2013, agriculture in south journal, 44 (5): 730-734.), design two special primers, carry out 3'RACE PCR.Gene Specific primer sequences is respectively, GSP1:5'-CACTACTAATCGTCAAGTCACCTTCTGT-3'(SEQ ID NO.3)；GSP2: 5'-CTTCTGTAAGAGGCGCAACGGCTT-3'(SEQ ID NO.4), the primer arranged in pairs or groups with it is that adapter-primer is AUAP: 5'-GGCCACGCGTCGACTAGTAC-3'(SEQ ID NO.5).Take the rare male flower of horse, carry out the extraction of RNA, with reference to BioTeke Generic plant total RNA extraction reagent box (centrifugal column type) is carried out.Take total serum IgE 1 μ g, cDNA synthesis and use PrimeScript RTase (Takara) reverse transcription, with Oligo d (T) the primer AP:5'-of belt lacing GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3'(SEQ ID NO.6) carry out reverse transcription, it is thus achieved that the first chain cDNA.With apocarya male flower the first chain cDNA as template, carrying out nest-type PRC, first round PCR reaction condition is: 94 DEG C 5min thermal denaturation；94 DEG C of 45s, 65 DEG C of 45s, 72 DEG C of 1min, totally 35 circulations；72 DEG C extend 10min.By first round PCR solution Dilute 10 times, in this, as template, carry out second and take turns PCR, the reaction condition same first round.Take turns PCR primer glue by second to reclaim After test kit (Genscript) reclaims, connect with pMD19-T carrier (Takara, Japan), then convert coliform DH5 α, choose Select positive colony, check order.Fragment order-checking obtained and original conservative fragments are compared, are spliced, it is thus achieved that sequence is long Degree is 1013bp.

In order to obtain its maximum open reading frame, design gene specific primer, VvAG-ORF sense:5'-at its two ends ATGGGGAGGGGGAGGATAGAAA-3'(SEQ ID NO.7) and VvAG-ORF antisense:5'- CGCCATAACAGGGCAATAACCT-3'(SEQ ID NO.8), with apocarya male flower the first chain cDNA as template, to enter Performing PCR expands, and PCR reaction condition is 94 DEG C of 5min thermal denaturations；94 DEG C of 45s, 48 DEG C of 45s, 72 DEG C of 1min, 35 circulations；72℃ Extend 10min, 4 DEG C of preservations, PCR primer is reclaimed, clones, checks order, after analyzing, obtain sequence shown in SEQ ID NO.1. The ORF of CiAG is 684bp, encodes 227 aminoacid, and aminoacid sequence is as shown in SEQ ID NO.2.

Embodiment 2 apocarya CiAG gene expression characteristic in Different Organs

As material, real-time fluorescence quantitative RT-PCR is utilized with three kinds of apocarya (Shaoxing, ripple Buddhist nun, Ma Han) Expression in apocarya different tissues blade, current-year branch, female flower, male flower, young fruit is studied.Each group Knitting the same step of the extraction () of total serum IgE, the synthesis of cDNA is with eliminating the test kit of residual DNA, PrimeScriopt in RNA RT reagent kit with gDNA Eraser (Takara, Japan), concrete steps reference reagent box description is carried out.In Ginseng use Semen Caryae Cathayensis actin gene (all Qin. the preliminary analysis [J] of gene expression between the Semen Caryae Cathayensis fruit period of maturation. Linan: Zhejiang Agricultural university, 2010), primer sequence is ACTIN-F:5'-GCTGAACGGGAAATTGTC-3'(SEQ ID NO.9) and, ACTIN- R:5'-AGAGATGGCTGGAAGAGG-3'(SEQ ID NO.10).Detection of expression primer is designed according to CiAG gene order VvAG qPCR sense:5'-AGGCTGCTACTCTACGACAAC-3'(SEQ ID NO.11), VvAG Qpcr Antisense:5'-GGTTCCTCTCATTCTCCGCTATC-3'(SEQ ID NO.12).With the cDNA from different tissues it is Template carries out real-time fluorescence quantitative RT-PCR analysis.

Interpretation of result shows (Fig. 1), and CiAG gene all has expression in three kinds, genitals (male flower, female flower, Young fruit) in expression be significantly higher than the expression in nutrition organs (blade, branch), illustrate that this gene rises in flower development process Important effect.

The Function Identification of embodiment 3CiAG transcription factor

Extract the T-carrier containing CiAG encoding gene purpose fragment and expression vector pCAMBIA1301 plasmid DNA, by limit Property restriction endonuclease Kpn I and Sac I double digestion processed, cuts glue and reclaims, and then connects with T4DNA ligase, converts escherichia coli.Extract matter Grain, PCR and enzyme action are identified.Escherichia coli plasmid freeze-thaw method proceeds to Agrobacterium (EHA105) competent cell, and bacterium solution PCR is reflected Determine positive colony.Obtain CiAG overexpression expression vector pCAMBIA1301-CiAG (Fig. 2).By Overexpression vector by using Freeze-thaw method pCAMBIA1301-CiAG is proceeded to Agrobacterium tumefaciems strain EHA105 (Avsian-Kretchmer et al, 2004, Plant Physiology, 135:1685-1696) in.The pCAMBIA1301-CiAG mediation by Agrobacterium strain EHA105, turns Change arabidopsis, utilize antibiotic-screening and round pcr screening positive transgenic plant.Total with the resistant plant through antibiotic-screening RNA is template, carries out RT-PCR reaction (Fig. 3) with non-transgenic Arabidopsis plants total serum IgE for negative control, and arabidopsis 18S makees Extract for RNA and cDNA synthesis successfully comparison.By the transgenic T2 that identifies for seed and wildtype Arabidopsis thaliana seed simultaneously Being multicast in cultivation matrix, culture conditions is: relative humidity 80%, intensity of illumination 80-200 μm ol/M2/S, temperature the photoperiod be 16h illumination, 22 DEG C/8h is dark, 17 DEG C.Being analyzed its phenotype, transgenic Arabidopsis plants downgrades (table 1), blooms than open country Raw type plant in advance, has bloomed when four basal leaves occur (Fig. 4).

Table 1

Strain	Highly (cm)
		WT	25.5
T1	9.3
		T2	8.9
T3	8.7

Claims (10)

1. an apocarya MADS-box class transcription factor CiAG, aminoacid sequence is as shown in SEQ ID NO.2.

2. the encoding gene of apocarya MADS-box class transcription factor CiAG described in claim 1.

Encoding gene the most according to claim 2, it is characterised in that described apocarya MADS-box class transcribe because of The cDNA gene nucleotide series of son is as shown in SEQ ID NO.1.

4. contain the expression vector of the encoding gene of apocarya MADS-box class transcription factor described in Claims 2 or 3.

Expression vector the most according to claim 4, it is characterised in that described expression vector is by described shell mountain core The encoding gene of Fructus Persicae MADS-box class transcription factor CiAG is inserted into Kpn I and Sac I restriction enzyme site of pCAMBIA1301 carrier Between gained.

6. contain the Host Strains of encoding gene described in Claims 2 or 3.

Host Strains the most according to claim 6, it is characterised in that described Host Strains is to be turned by pCAMBIA1301-CiAG Enter Agrobacterium tumefaciems EHA105 gained.

8. apocarya MADS-box class transcription factor CiAG described in claim 1 is in cultivating early blossoming dwarfing arabidopsis Application.

9. the encoding gene of apocarya MADS-box class transcription factor CiAG described in Claims 2 or 3 is being cultivated early Flower downgrades the application in arabidopsis.

10. the application in cultivating early blossoming dwarfing arabidopsis of the expression vector described in claim 4 or 5.