CN104004076A - Thin-shell hickory nut MADS-box transcription factor CiAG as well as encoding gene and application thereof - Google Patents

Abstract

The invention discloses a thin-shell hickory nut MADS-box transcription factor CiAG as well as an encoding gene and application thereof. The thin-shell hickory nut MADS-box transcription factor CiAG is a protein having the amino acid sequence shown in SEQ ID NO. 2 of the sequence table and the encoding gene of the transcription factor CiAG is a DNA sequence in SEQ ID NO. 1 of the CiAG gene. The thin-shell hickory nut MADS-box transcription factor CiAG can be used for breeding early-flowering and dwarf varieties of plants. The CiAG is a specifically expressed gene, and the expression patterns of the CiAG is associated with tissue and developmental stage of the plant. Transgenic Arabidopsis overexpressed by the CiAG, compared with a control group, blooms early than the wild-type plants, and is blossomed when four pieces of basal leaves appear, and furthermore, the plants exhibit the dwarf characteristics, which shows that CiAG can control flowering transition of the plants, affect the flowering period and vegetative growth of the plants and further regulate the reproductive growth of the plants.

Description

A kind of apocarya MADS-box class transcription factor CiAG and encoding gene and 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 and application.

Background technology

The virgin phase of apocarya is very long, needs about 15 years, and male flower grows early, female flower development evening.In addition, apocarya is the different flowering plant of monoecism, has female antetype, male antetype and three kinds of different types of male and female homotype, and male flower main raw on the male inflorescence of raw branch the previous year middle and lower part, and once raceme of female flower the raw top at fruitful branch.As can be seen here, the growth of apocarya Male and female flowers has unique feature, its flower development gene is studied, for cultivating early blossoming real kind morning and having great importance shortening the juvenile phase.

MADS-box is a DNA binding domains that conservative property is very strong, in yeast Mini Chromosome Maintenance1 (MCM1), Arabidopis thaliana AGAMOUS (AG), Common Snapdragon DEFICIENS (DEF) and serum human response factors Serum ResponseFactor (SRF), find, 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 and there is not yet report in dwarfing fruit trees.

Summary of the invention

Goal of the invention:

The object of this invention is to provide a kind of apocarya MADS-box class transcription factor CiAG.

Another object of the present invention is to provide the encoding gene of this transcription factor CiAG.

Another object of the present invention is to provide the application of this transcription factor.

Object 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, called after CiAG, derive from apocarya (Carya illinoensis (Wangench.) K.Koch) improved seeds ' Ma Han ', aminoacid sequence is as shown in SEQ ID NO.2.

The encoding gene of apocarya MADS-box class transcription factor of the present invention, its cDNA sequence is as shown in SEQ ID NO.1; In sequence, contain the maximum open reading frame of 684bp, 227 amino acid residue sequences shown in coding SEQ ID NO.2.

The expression vector that contains CiAG genes of SEQ ID NO.1 of the present invention.

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

Host Strains refers to the agrobacterium tumefaciens EHA105. that pCAMBIA1301-CiAG is proceeded to

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 relating 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, contain encoding gene expression vector in the application of cultivating in 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 specific expressed in male flower, female flower and young fruit.Overexpression CiAG encoding gene in transgenic arabidopsis, compared with control group, significantly shift to an earlier date flowering period, and plant is downgraded.The result shows that CiAG gene can control one-tenth China transition process of plant, can make plant downgrade simultaneously.

Particularly the precocious apocarya of downgrading of early blossoming is significant for cultivating early blossoming, dwarf plant kind for CiAG of the present invention, aspect crop breeding, is with a wide range of applications.

Utilize plant expression vector of the present invention, in CiAG gene transfered plant body, the transfer-gen plant that can obtain blooms shifts to an earlier date, plant is downgraded.

Brief description of the drawings

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 Arabidopis thaliana and control group RT-PCR and detect.

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

Fig. 4 turns the Phenotypic Observation of CiAG gene Arabidopis thaliana and contrast

WT: wild-type; T: transgenic line

Embodiment

Methods all in following example, without special instruction, are ordinary method

The cloning and identification of embodiment 1 apocarya CiAG gene

Experiment material is apocarya kind ' Ma Han ', has cloned the conservative fragments (Mo Zhenghai etc., 2013 that obtain according to this laboratory, agriculture in south journal, 44 (5): 730-734.), design two special primers, carry out 3'RACE PCR.Gene specific primer sequence is respectively, GSP1:5'-CACTACTAATCGTCAAGTCACCTTCTGT-3'(SEQ ID NO.3); GSP2:5'-CTTCTGTAAGAGGCGCAACGGCTT-3'(SEQ ID NO.4), with the primer of its collocation be that joint primer is AUAP:5'-GGCCACGCGTCGACTAGTAC-3'(SEQ ID NO.5).Get the rare male flower of horse, carry out the extraction of RNA, carry out with reference to the general plant total RNA extraction reagent of BioTeke box (centrifugal column type).Get total RNA1 μ g, synthetic PrimeScript RTase (Takara) ThermoScript II that adopts of cDNA, with Oligo d (T) the primer AP:5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3'(SEQ ID NO.6 of belt lacing) carry out reverse transcription, obtain the first chain cDNA.Taking apocarya male flower the first chain cDNA as template, carry out nest-type PRC, first round PCR reaction conditions is: 94 DEG C of 5min thermally denatures; 94 DEG C of 45s, 65 DEG C of 45s, 72 DEG C of 1min, totally 35 circulations; 72 DEG C are extended 10min.By 10 times of first round PCR solution dilutions, using this as template, carry out second and take turns PCR, the same first round of reaction conditions.Take turns PCR product glue by second and reclaim after test kit (Genscript) recovery, connect with pMD19-T carrier (Takara, Japan), then transform colibacillus DH5 α, select positive colony, check order.The fragment that order-checking is obtained and original conservative fragments are compared, are spliced, and acquisition sequence length is 1013bp.

In order to obtain its maximum open reading frame, at its two ends design gene specific primer, VvAG-ORF sense:5'-ATGGGGAGGGGGAGGATAGAAA-3'(SEQ ID NO.7) and VvAG-ORF antisense:5'-CGCCATAACAGGGCAATAACCT-3'(SEQ ID NO.8), with taking apocarya male flower the first chain cDNA as template, carry out pcr amplification, PCR reaction conditions is 94 DEG C of 5min thermally denatures; 94 DEG C of 45s, 48 DEG C of 45s, 72 DEG C of 1min, 35 circulations; 72 DEG C are extended 10min, and 4 DEG C of preservations are reclaimed PCR product, clone, check order, and obtain afterwards by analysis sequence shown in SEQ ID NO.1.The ORF of CiAG is 684bp, 227 amino acid of encoding, and aminoacid sequence is as shown in SEQ ID NO.2.

The expression characteristic of embodiment 2 apocarya CiAG genes in Different Organs

Taking three kinds of apocarya (Shaoxing, ripple Buddhist nun, Ma Han) as material, utilize real-time fluorescence quantitative RT-PCR to be studied the expression in apocarya different tissues blade, current-year branch, female flower, male flower, young fruit.The same step of extraction (one) of each total tissue RNA, the synthetic use of cDNA can be eliminated the test kit of residual DNA in RNA, PrimeScriopt RT reagent kit with gDNA Eraser (Takara, Japan), concrete steps reference reagent box specification sheets carries out.Internal reference use Semen Caryae Cathayensis actin gene (all Qin. the initial analysis [J] of Semen Caryae Cathayensis fruit ripening period genetic expression. Linan: Zhejiang A & F University, 2010), primer sequence is ACTIN-F:5'-GCTGAACGGGAAATTGTC-3'(SEQ ID NO.9), ACTIN-R:5'-AGAGATGGCTGGAAGAGG-3'(SEQ ID NO.10).According to CiAG gene order design detection of expression primer VvAG – qPCR sense:5'-AGGCTGCTACTCTACGACAAC-3'(SEQ ID NO.11), VvAG – Qpcr antisense:5'-GGTTCCTCTCATTCTCCGCTATC-3'(SEQ ID NO.12).Carry out real-time fluorescence quantitative RT-PCR analysis taking the cDNA from different tissues as template.

Interpretation of result shows (Fig. 1), CiAG gene all has expression in three kinds, expression amount in reproductive organ (male flower, female flower, young fruit) is significantly higher than the expression amount in vegetative organ (blade, branch), illustrates that this gene plays an important role in flower development process.

The Function Identification of embodiment 3CiAG transcription factor

The T-carrier that extraction contains CiAG encoding gene object fragment and expression vector pCAMBIA1301 plasmid DNA, with restriction enzyme Kpn I and Sac I double digestion, cut glue and reclaim, and then connects with T4DNA ligase enzyme, transforms intestinal bacteria.Extract plasmid, PCR and enzyme are cut qualification.Escherichia coli plasmid is proceeded to Agrobacterium (EHA105) competent cell with freeze-thaw method, and bacterium liquid PCR identifies positive colony.Obtain CiAG overexpression expression vector pCAMBIA1301-CiAG (Fig. 2).By Overexpression vector by pCAMBIA1301-CiAG being proceeded in agrobacterium tumefaciens strain EHA105 (Avsian-Kretchmer et al, 2004, Plant Physiology, 135:1685-1696) with freeze-thaw method.PCAMBIA1301-CiAG is by the mediation of Agrobacterium strain EHA105, and arabidopsis thaliana transformation, utilizes antibiotic-screening and round pcr to screen positive transfer-gen plant.Taking the total RNA of the resistant plant through antibiotic-screening as template, carry out RT-PCR reaction (Fig. 3) with the negative contrast of the total RNA of transgenic arabidopsis plant not, Arabidopis thaliana 18S extracts as RNA and cDNA is synthetic successfully contrasts.The transgenosis T2 identifying is broadcast in cultivation matrix for seed and wild-type Arabidopis thaliana seed simultaneously, and culture conditions is: relative humidity 80%, and intensity of illumination 80-200 μ mol/M2/S, the temperature photoperiod is 16h illumination, 22 DEG C/8h dark, 17 DEG C.Its phenotype is analyzed, and transgenic arabidopsis plant is downgraded (table 1), blooms than wild-type plant in advance, has bloomed (Fig. 4) in the time that four basal leaves occur.

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 claimed in claim 1.

3. encoding gene according to claim 2, is characterized in that the cDNA gene nucleotide series of described apocarya MADS-box class transcription factor is as shown in SEQ ID NO.1.

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

5. expression vector according to claim 4, is characterized in that described expression vector is that the encoding gene of described apocarya MADS-box class transcription factor CiAG is inserted into gained between the Kpn I of pCAMBIA1301 carrier and Sac I restriction enzyme site.

6. contain the Host Strains of gene described in claim 2 and 3.

7. Host Strains according to claim 6, is characterized in that described Host Strains is that pCAMBIA1301-CiAG is proceeded to agrobacterium tumefaciens EHA105 gained.

8. apocarya MADS-box class transcription factor CiAG claimed in claim 1 is in the application of cultivating in early blossoming dwarf plant.

9. the encoding gene of the apocarya MADS-box class transcription factor CiAG described in claim 2 or 3 is in the application of cultivating in early blossoming dwarf plant.

10. the expression vector described in claim 4 or 5 is in the application of cultivating in early blossoming dwarf plant.