CN107840872B

CN107840872B - Albumen and the application of wax plum CpWOX13 gene and its coding

Info

Publication number: CN107840872B
Application number: CN201710927919.9A
Authority: CN
Inventors: 李志能; 江英杰; 李先源; 眭顺照; 李名扬
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2017-08-16
Filing date: 2017-10-09
Publication date: 2019-06-21
Anticipated expiration: 2037-10-09
Also published as: CN107840872A

Abstract

The albumen and its application in florescence control and raising biomass that the present invention provides wax plum CpWOX13 gene and its coding.The amino acid sequence of wax plum CpWOX13 provided by the invention encodes the gene order of the albumen as shown in SEQ ID No.2 as shown in SEQ ID No.1.The present invention clones for the first time obtains wax plum CpWOX13 gene, and pass through transgenic technology, the function of the gene is demonstrated in plant (arabidopsis), transgenic arabidopsis is compared with wild type, advance flowering period, plant height are got higher, root system is elongated, lateral root increases, further to improve ornamental plant, as the florescence control of wax plum and increase species biomass offer may.

Description

Albumen and the application of wax plum CpWOX13 gene and its coding

Technical field

The present invention relates to genetic engineering fields, specifically, being related to wax plum CpWOX13 gene and its in florescence control Using.

Background technique

Wax plum [Chimonanthus praecox (L.) Link.] also cry wintersweet, is also known as incense wood, Tang Mei, rock Zisang Deng being the machaka of Laurales Calycanthaceae wax-cakes bait, belong to second level endangered plants (left Dan Dan etc., 2007).Originate in China head River downstream, is distributed mainly on the ground such as Soviet Union, Zhejiang, Anhui, Hubei Province, Jiangxi, and artificial cultivation history can be traced to the Tang Dynasty, Winter blooming and resists cold resistance to Drought has original ornamental value and considerable economic value.

Calycanthaceae is worldwide divided into 4 categories, 10 kinds (Zhang Ruohui, 1998).This four categories are respectively Positioned at the chair Pterostyrax (Idiospermun Blake) of Queensland ,Australia, southeastern US and California growth Calycanthus (Calycantus L.), further include the prunellae vulgaris L (Sinocalycanthus in temperate zone originating in China ) and wax-cakes bait (Calycantus L.) Cheng.Li Ye (1998) by study wax plum respectively belong between evolution and development relationship, Calycanthaceae has been divided into two subfamilies again, has been chair tree subfamily (Idiospermoideae) and wax Prunoideae respectively (Calycanthoideae), wherein wax Prunoideae includes prunellae vulgaris L, wax-cakes bait and Calycanthus, and chair tree subfamily is only There is chair Pterostyrax.The horizontal highest category of evolution is chair Pterostyrax in four categories, and the category of most original is wax-cakes bait.

With the development of modern molecular biology, biotechnology has been widely used for grinding for plant, animal and microorganism In studying carefully.Molecular biology research in relation to wax plum is broadly divided into assortment and gene function two parts.RAPD, ISSR etc. at present Molecular marking technique is expertly for the assortment of wax plum and the analysis of gregarious genetic structure etc..Du Lingjuan (2012) et al. is adopted The Cultivars Natural Populations hereditary variation of In Nanjing 38 is analyzed with RAPD technology, find group in each single plant it Between hereditary difference it is bigger, and the genetic distance between population is closer, therefore show that hereditary variation takes place mostly in gregarious, this It is consistent with morphological classification result.The genome of wax plum climax leaves and tender leaf is extracted inspection respectively by Zhao Mingxiao etc. (2011) Survey is compared, and is classified using AFLP technology to Cultivars for descendant and Natural Populations analysis of genetic diversity has carried out place mat.And Zhao Bing et al. establishes the AFLP molecular labeling system in relation to wax plum, more for studying the systematic growth of wax plum and heredity for AFLP technology Sample lays the foundation (Zhao Bing, 2007)；Yang Jia (2012) uses EST-SSR technology to the genetic structure and genetic diversity of wax plum Property is analyzed, and has carried out Primary Study to the genetic structure feature and level of genetic diversity of wax plum.

Gene in addition, Sui is along constructing first wintersweet immature flower cDNA library according to (2006), and during blooming to wax plum Expression analysis has been carried out, has successively cloned the relevant gene of different function on its basis, and systematic research has been carried out to it； If Cpcor413pm1 (Qin Hua, 2006) is related to the winter resistance of wax plum, it is transferred to tobacco, transgene tobacco is handled It was found that the low temperature tolerance ability of transgene tobacco improves；CpAP3 forms related with petal-shaped allelotaxis；CpPEX22 and anti-aging, It is anti-oxidant that there are certain relationships；CpAGL6 is transferred in arabidopsis, it is short to occur Blooming, plant compared with wildtype Arabidopsis thaliana Change, the form of stamen and number are all changed；CpKTI trypsase suppressor is transferred to tobacco overexpression, hair The insect resistace of existing transgene tobacco improves；Cpchia (Xie Shuzhang, 2009) chitinase gene；CpPR-4 (Qin Dynasty, 2012) disease Journey related protein gene etc..The Primary Study of above-mentioned wax plum gene function is made centainly for later wax plum molecular biology research Contribution.

The WOX gene reported in recent years being found from plant without exception, so generally believing that WOX is that plant is peculiar Transcription factor, and being all classified as WOX (WUSHOMEOBOX) transcription factor family with the transcription factor of WUS box in plant. 15 members (WUS, WOX1-WOX14) such as in arabidopsis (Arabidopsis thaliana) take part in the hair of embryo respectively It educates, the formation of floral organ, flower development, the weight such as the development of lateral organ and the maintenance of stem apex and tip of a root apical meristem area stem cell Want vital movement.There are three clade for WOX albumen in frame of reference development tree arabidopsis, are respectively as follows: WUS clade (WUS Clade), i.e. modern evolution branch, including WUS, WOX1, WOX2, WOX3, WOX4, WOX5, WOX6, WOX7 and they in seed Ortholog protein in plant；Intermediate clade (Intermediate Clade), is sister's clade of WUS clade, Including WOX8, WOX9, WOX11, WOX12 and their ortholog proteins in vascular plant；Ancient clade (Ancient Clade), including WOX10, WOX13 and WOX14 and they dimension pipe and non-vascular plant in ortholog egg White (Hao et al., 2013；Zhang et al.,2011).

WUSCHEL-related homeobox13 (WOX13) gene is member (the Prigge et of WOX gene family Al., 2005), belong to ancient clade, in arabidopsis participate at flower conversion and root growth course, also with the hair of lotus throne leaf Pass is given birth to, the regulation of development of floral organs process is additionally participated in.This kind of transcription factors participate in the regulation of plant stem cell. Laux in 1996 has found earliest and has determined WUS gene function (Prigge et al., 2005) that he has found true leaf in mutant Missing and the disappearance of apical dominance be positively correlated, the missing of WUS gene causes seed speed of germinating slack-off, and hypoevolutism is even It stagnates, branch quantity and density are in rising trend, and stem situation is unobvious, and branch repeats the development models of stem lotus throne leaf. Successful clone goes out to encode the WUS gene of 291 amino acid, and analyzing its topology discovery WUS is in homeodomain albumen One new subtype (Haecker et al., 1991).Hereafter gradually go deep into about the research of WOX family gene, systematically formed The basic classification systems of three big branches.

Wax plum is as rare Winter blooming plants, and the fragrance of flowers is strong, has higher economy and ornamental value.Therefore to it Flower development research is very significant.And in arabidopsis WOX13 participate at flower conversion and root growth course, also with the hair of lotus throne leaf Pass is given birth to, the regulation of development of floral organs process is additionally participated in, so, can be to CpWOX13 gene function analysis in wax plum Further research wax plum blooms and the regulatory mechanism of biomass provides some valuable references, and mentions for plant molecular breeding For genetic resources.

Summary of the invention

The object of the present invention is to provide wax plum CpWOX13 gene and its applications in florescence and biomass regulation.

In order to achieve the object of the present invention, wax plum CpWOX13 albumen of the invention, are as follows:

1) protein that the amino acid shown in SEQ ID No.2 forms；Or

2) be substituted, lack or add in the amino acid sequence shown in SEQ ID No.2 one or several amino acid and With the same active protein as derived from 1).

The present invention also provides the encoding genes of wax plum CpWOX13 albumen, the i.e. cDNA sequence of CpWOX13 gene such as SEQ ID Shown in No.2.

The present invention also provides the carrier containing CpWOX13 gene, host cell and engineering bacterias.

The present invention also provides CpWOX13 genes in the interim application with raising biomass of regulation plant flowers.The plant is Arabidopsis.

The present invention also provides application of the CpWOX13 gene in prepare transgenosis plant.

The present invention further provides a kind of construction methods of transgenic plant will be contained using the method for mediated by agriculture bacillus The carrier of CpWOX13 gene is transferred in plant tissue, screening transgenic plant.

The invention has the following advantages that

The present invention clones for the first time obtains wax plum CpWOX13 gene, and by transgenic technology, tests in plant (arabidopsis) Demonstrate,prove the function of the gene, the results showed that, transgenic arabidopsis advance flowering period, plant height are got higher, root system is elongated, lateral root increases, and are Ornamental plant is further improved, as the florescence control of wax plum and increase species biomass offer may.

Detailed description of the invention

Fig. 1 is wax plum CpWOX13 protein sequence phylogenetic tree, wherein arabidopsis (Arabidopsis thaliana) WOX14A (NP_001322715), WOX14B (NP_173493), WOX10 (NP_173494), WOX13 (NP_195280), WOX11A (NP_187016), WOX11B (AAP37140), WOX12A (NP_197283), WOX12B (NP_001330983), WOX7A (NP_196196), WOX7B (AIR72305), WOX7C (OAO92889), WOX5 (NP_187735), WOX4A (NP_ 001320246), WOX4B (NP_175145), WOX1A (NP_188428), WOX1B (AAP37133), WOX8 (NP_199410), WUS (CAA09986), WOX2B (AAP37131), the WOX8 (XP_ of WOX2A (OAO90278), Erythranthe guttata 012845314)；The WOX2 (XP_015639083) of Oryza sativa Japonica Group, WOX8A (XP_ 015626455), WOX12 (XP_015638690) WOX4 (XP_015635367), WOX5 (XP_015644226)；Oryza The WOX3-like (XP_015692532) of brachyantha, WOX5-like (XP_006646469)；Phoenix The WOX8-like (XP_008812362) of dactylifera；The WUS (AAP37131) of Nelumbo nucifera, WOX3- Like (XP_010259966), WOX1-like (XP_010274717), WOX2 (XP_010260182), WOX8-like (XP_ 010270477), WOX9-like (XP_010279211), WOX11 (XP_019054443), WOX7-like (XP_ 010256620), WOX5-like (XP_010262794), WOX4-like (XP_010270556), Vitis vinifera's The WOX8 (XP_006855728) of WOX8 (XP_002272420), Amborella trichopoda；Picea abies's WOX8C (AGL53586), WOX8D (AGL53587), WOX8B (AGL53585), WOX8A (AGL53584).

Fig. 2 show CpWOX13 gene in the different month inflorescence samples of wax plum Real-time PCR Analysis as a result, Actin Gene is as internal reference.Wherein, 4IM, 5FM, 7FM and 9FM difference 4,5,7, the inflorescence of September part.

Fig. 3 show 35S::CpWOX13 overexpression conversion wildtype Arabidopsis thaliana (Col-0) T2 for (14 is small in the long-day Shi Guangzhao/10 hour dark) under the conditions of real-time quantitative PCR testing result；CpWOX13 relative expression quantity.Three repetitions, Actin As internal reference.Wherein, WT: wildtype Arabidopsis thaliana；Other are quasi- for overexpression 35S::CpWOX13/1#, 2#, 3#, 6#, 7#, 8# Southern mustard transgenic line.Reference gene Actin, if 3 technologies repeat.

Fig. 4 is that 35S::CpWOX13 overexpression converts wildtype Arabidopsis thaliana (Col-0) T2 generation in long-day in (the 14 small time According to/10 hours dark) under the conditions of, arabidopsis endogenous gene real-time quantitative PCR testing result；Three repetitions, Tubulin be used as in Ginseng.Wherein, the 6# of the 8# of CpWOX1 gene high expression, the 3# of medium expression and low expression are to indicate variable expression in Fig. 3 35S::CpWOX13 transgenic line, carry on subsequent Phenotypic Observation and endogenous gene quantitative analytical experiment.

Fig. 5 is that 35S::CpWOX13 overexpression converts the florescence in wildtype Arabidopsis thaliana (Col-0) T2 generation, plant height isophenous Analyze result.A, B is that 28d wildtype Arabidopsis thaliana is buddingged, and is followed successively by WT, strain 6#, strain 3#, strain 8# from left to right；C, D is Degree of contrast when 60d wildtype Arabidopsis thaliana knot last fruit pod, be followed successively by C from left to right strain 6#, strain 3#, strain 8# and Right side is WT in WT, D；E, F is that the transgenic plant bloomed earliest in strain 3# and strain 8# and WT are compared, left side WT；Institute It is wormcast with soil: vermiculite: fertile soil=2:2:1；Condition of culture is long-day LD:16h illumination/8h dark；21 DEG C of temperature Left and right.

Fig. 6 is the root growth phenotype point that 35S::CpWOX13 overexpression converts wildtype Arabidopsis thaliana (Col-0) T2 generation Analyse result.Wherein, left side is transgenic plant, right side WT, transgenic plant lateral root number and intensive journey in red round frame in A Degree is apparently higher than WT；The front that B is A is shone, left side WT；C, red boxes show WT in D, other are that different strains turn base Because of plant, it is seen that WT root long is obviously partially short；Culture medium is MS non-resistant culture medium

Specific embodiment

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment According to conventional laboratory conditions, such as Sambrook molecular cloning experiment handbook (Sambrook J&Russell DW, Molecular cloning:a laboratory manual, 2001), or according to the condition of manufacturer's specification suggestion.

The clone of 1 wax plum CpWOX13 gene of embodiment and expression analysis

1 wax plum CpWOX13 gene cloning

The extraction of 1.1 total serum IgEs

(1) taking a mouthful wax plum (Chimonanthus praecox) of using up, (plantation is big in It In Beibei, Chongqing day No. 2 southwest of the means of livelihood In school garden beside the 4th sports ground, conventional maintenance management) it bud, bud (4,5,7, September part), blooms, decay and blade is mixed Close sample.RNA extracts glassware, mortar, pestle, scissors, tweezers, medicine spoon used etc. and is existed with 0.1%DEPC aqueous solution Then it is spare to be wrapped in 180 DEG C of baking ovens cooling after hot air sterilization 4h with tinfoil by immersion treatment 12h at 37 DEG C.

RNA extracts the RNAprep Pure plant total RNA extraction reagent box using the production of Beijing Tiangeng company, extraction process It is slightly changed according to specification, concrete operations are as follows:

(1) it takes 500 μ L lysate RL to be transferred in 1.5mL centrifuge tube, 1% beta -mercaptoethanol is added, mix spare.In liquid nitrogen After appropriate plant tissue is ground into fine powder, 50mg fine powder is taken to be transferred in centrifuge tube, above-mentioned 500 μ L of RL is added, immediately with the vibration that is vortexed It swings device and acutely shakes mixing, crack it sufficiently.

(2) solution is centrifuged 5min in 12,000rpm, the careful supernatant drawn in collecting pipe to new RNase-free from In heart pipe, suction nozzle avoids contact with the pellet cell debris in collecting pipe as far as possible.

(3) it is slowly added to the dehydrated alcohol (usually 250 μ L) of 0.5 times of supernatant volume, (it is heavy to be likely to occur at this time for mixing Form sediment), obtained solution and precipitating are transferred to together in adsorption column CR3,12,000rpm centrifugation 30-60s are outwelled in collecting pipe Waste liquid puts back to adsorption column CR3 in collecting pipe.

(4) preparation of DNase I working solution: 10 μ L DNase I storing liquids is taken to be put into new RNase-free centrifuge tube In, 70 μ L RDD solution are added, it is soft to mix.

(5) 80 μ L DNase I working solutions are added to the center adsorption column CR3, are placed at room temperature for 15min.

(6) 350 μ L protein liquid removal RW1,12,000rpm centrifugation 30-60s are added to the center adsorption column CR3 and outwell collecting pipe In waste liquid, adsorption column CR3 is put back in collecting pipe.

(7) 500 μ L rinsing liquid RW (using preceding addition dehydrated alcohol) is added to the center adsorption column CR3, is stored at room temperature 2min, 12,000rpm centrifugation 30-60s, outwell the waste liquid in collecting pipe, adsorption column CR3 are put back in collecting pipe.Repeat the step 1 It is secondary.

(8) 12,000rpm are centrifuged 2min, outwell waste liquid.Adsorption column CR3 is placed in and is placed at room temperature for several minutes, thoroughly to dry in the air Remaining rinsing liquid in dry adsorbent material.

(9) adsorption column CR3 is put into a new RNase-free centrifuge tube, is vacantly dripped to the intermediate position of adsorbed film Add 20-100 μ L RNase-free ddH2O, be placed at room temperature for 2min, 12,000rpm centrifugation 2min obtain RNA solution.

The synthesis of 1.2 wax plum the first chains of cDNA

The conjunction of the first chain of cDNA is carried out using the PrimeScript RT-PCR Kit reverse transcription reagent box of TaKaRa company At.To specifications the step of carries out, in total in two steps: the first step is removal genomic DNA reaction；Second step is that reverse transcription is anti- It answers.Concrete operations are as follows:

(1) according to the form below prepares first step reaction system:

1 the first step system of reverse transcription of table

(2) 42 DEG C of reaction 5min are mixed liquor 1.

(3) according to the form below configures second step reaction system:

2 the second step system of reverse transcription of table

(4) reverse transcription program is 37 DEG C of reaction 15min, then 85 DEG C of reaction 5sec；

(5) cDNA product is stored in -20 DEG C of refrigerators.

1.3 CpWOX13 gene clonings

According to the wintersweet immature flower cDNA library annotation information (Liu at al., 2014) of this laboratory building, detection is wrapped The cDNA sequence of CpWOX13 gene containing complete ORF frame.The cDNA library experimental material is the single sonorous stone mouth wax plum planted in the school, is taken The flower of its different times is material building.Specific primer is designed according to gained cDNA sequence, upstream and downstream primer includes complete ORF sequence, primer sequence such as table 3:

3 wax plum CpWOX13 gene cloning primer of table

Note: R:A/G；S:G/C；Y:C/T；K:G/T；W:A/T；F represents upstream after primer, and R represents downstream.

The purifying of 1.4 PCR recovery products

The product that PCR amplification obtains is detected with the Ago-Gel of l%, is 800- by size in amplified production Band and specific band between 1000bp scale off, and are recycled, and return according to Tiangeng company plain agar sugar gel DNA glue Kit specification is received to recycle target DNA fragment.

The connection of 1.5 PCR recovery products

Coupled reaction system is shown in Table 4.

4 coupled reaction system of table

PCR recovery product	1-3μl
		Buffer solution Solution I	5μl
PMD19-T vector	0.5μl
		RNase-free ddH2O	1.5-3.5μl
Total	10μl

4 DEG C overnight, are attached reaction.

1.6 CaCl₂Method prepares bacillus coli DH 5 alpha competent cell

(1) it from one single colonie of picking on the E. coli plate newly activated, is inoculated in 3-5mL LB Liquid Culture, 37 DEG C Shaken cultivation 12h or so, until logarithmic growth phase.The bacteria suspension is transferred with 1:100-1:50 in 50mL LB liquid medium In, 37 DEG C of oscillations expand culture, until stopping culture when OD600 is 0.4-0.6.

(2) culture solution is poured into the sterile 50mL centrifuge tube being pre-chilled on ice, places 10min on ice, in 4 DEG C, 4, 100rpm is centrifuged 10min.

(3) evacuation supernatant culture solution, is inverted 1min on aseptic filter paper, the 0.1M CaCl2 solution being pre-chilled on ice with 30mL Gently suspension cell, 4 DEG C, 4,100rpm, it is centrifuged 10min.

(4) liquid is discarded supernatant, the 0.1mo1/L CaCl2 solution that 2mL is pre-chilled on ice is added, gently suspension cell.It puts on ice It sets in a moment, that is, competent cell suspension has been made；The competent cell suspension prepared can be directly used for transformation experiment, can also Addition accounts for the glycerol that 15% or so high pressure sterilization of total volume is crossed, and is poured after mixing packing with liquid nitrogen after freezing rapidly thereon in -80 DEG C save.

The conversion of 1.7 connection products

Connection product converts competent escherichia coli cell

(1) E. coli competent is taken out from -80 DEG C of refrigerators, is placed on ice to melt.By connection product in 70 DEG C of water-baths 10min is inactivated in pot.

(2) by connection product or plasmid to be transformed, 100ng plasmid purification is added according to every 200 μ L competent cell Standard, be added in competent cell, stand 30min after mixing gently on ice.

Heat shock 90s in (3) 42 DEG C of water-baths is immediately placed on 2min on ice after taking out rapidly.

(4) the nonreactive LB liquid mediums of 37 DEG C of 600 μ L preheatings, in 37 DEG C after mixing, 130rpm shake culture are added 1h。

(5) 10,000rpm is centrifuged 1min at room temperature, discards 400 μ L supernatants, remaining 200 μ L bacterium night is resuspended, with nothing Resuspended bacterium solution is evenly coated on the LB solid plate containing corresponding antibiotic by the painting stick of bacterium, and 12-14h is cultivated in 37 DEG C of inversions.

1.8 positive colony cultures and PCR are identified

Monoclonal is screened by IPTG/X-GAL, takes sterile 1.5mL centrifuge tube, is added 0.5mL Amp's containing 50mg/L LB liquid medium, with sterile toothpick, gently picking single colonie is placed in centrifuge tube from transformed plate, 37 DEG C of 200rpm Shaken cultivation 8-10h in shaking table carries out PCR identification using it as template after bacterium solution is muddy.

Bacterium solution PCR amplification system:

PCR response procedures:

94 DEG C of denaturation 5min；94 DEG C of denaturation 30s, 54 DEG C of annealing 30s, 72 DEG C of extension 1min, 26 recycle；72 DEG C of extensions 10min。

1.9 sequencings and sequence analysis

Selection Insert Fragment size is sequenced with expected close and special positive colony.Wax plum CpWOX13 gene CDNA sequence encodes the amino acid sequence of albumen as shown in SEQ ID No.2 as shown in SEQ ID No.1.

By its homologous sequence that MEGA5 obtains the CpWOX13 protein sequence encoded and comparison carry out analysis construct into Change tree (Fig. 1), using NJ method, inspection parameter is set as 1000bootsrap repetition.As shown, all protein sequences are divided For apparent three groups, three clade of WOX family are respectively corresponded, study report unanimously with forefathers, wherein CpWOX13 is encoded Albumen and arabidopsis WOX13 albumen, grape WOX8 albumen belong to ancient clade, illustrate that its evolutionary degree is relatively low.? Sequence analysis is carried out with amino acid sequence of the Blastp to CpWOX13 in NCBI, as the result is shown itself and various plants WOX13 egg White amino acid sequence have compared with high homology, such as with apple (Malus domestica, XP_008392426.1), plum (Prunus mume, XP_008237971.1), peach (Prunus persica, ONI05494.1), soybean (Glycine max, XP_003523048.1 similitude) is 86%, wherein the phase with lotus (Nelumbo nucifera, XP_010270477.1) Like property highest, up to 94%.

2 wax plum CpWOX13 gene expression analysis

To wax plum CpWOX13 gene in the inflorescences primordium or inflorescence in different months expression analysis, find the gene in April There is expression in (4IM), May (5IM), July (7FM) and September (9FM), but expression has larger difference (Fig. 2).The gene Be 6.5 times of expression quantity in 4IM in 5IM period expression quantity highest, be 3.8 times and 3.3 times of 7FM and 9FM respectively, and 7FM and 9FM expression quantity difference is less (Fig. 2).

The wax plum CpWOX13 genetic transformation (arabidopsis) of 2 mediated by agriculture bacillus of embodiment

The building of 1 expression vector

According to requirement of experiment, the polyclone enzyme enzyme site characteristic on carrier pCAMBIA2301g is utilized in the present embodiment, It will be connected in expression vector after cDNA segment digestion on cloning vector.

The preparation of 2 Agrobacterium GV3101 and EHA105 electrization competent cells and the conversion of expression vector

(1) fresh Agrobacterium GV3101 and the EHA105 single bacterium of picking is fallen in 10mL LB liquid medium, 28 DEG C of oscillations It cultivates to late log phase；

(2) 1mL bacterium solution is taken to be added in the fresh LB liquid medium of 100mL, 28 DEG C of shaken cultivations to OD₆₀₀About 0.5- 1.0；

(3) bacterium solution is transferred in 50mL centrifuge tube, ice bath 30 minutes；

(4) 4 DEG C, 4000rpm is centrifuged 10 minutes, collects thallus, abandons supernatant；

(5) it precipitates and is resuspended with the HEPES sterile purified water that 50mL is pre-chilled；

(6) 4 DEG C, 4000rpm is centrifuged 10 minutes, collects thallus, abandons supernatant；

(7) twice, each HEPES halves for repetitive operation (5), (6)；

(8) thallus is resuspended with the sterile purified water containing 10% glycerol, dispenses by every 50 μ l volume of pipe to 1.5ml In Eppendorf pipe, as Agrobacterium competent cell is directly saved backup using or in -70 DEG C after packing；

(9) competent cell of the 50 above-mentioned preparations of μ l is taken, 0.5 μ g plasmid is added, mixes gently, is placed on ice；

(10) 2500 volts of electric shock 5ms, are added immediately 500 μ l LB liquid mediums；

(11) 28 DEG C, 150rpm, 4 hours of renewal cultivation；

(12) suitable cell is coated in screening LB solid medium tablets, and super-clean bench dries up surface layer liquid, 28 DEG C of trainings It supports 2-3 days.

The transformation of Arabidopsis thaliana (bud infusion method) of 3 mediated by agriculture bacillus

When arabidopsis floral bolting about 1cm high, main inflorescence is cut off.The raw inflorescence in side is issued to plant, and grows to bud When undeployed, transformation experiment is carried out.Be inoculated with the Agrobacterium colonies containing expression vector in 5mL LB liquid medium (Gen: 50mg/L, Kan:50mg/L) in, 28 DEG C, shaken cultivation is stayed overnight in 200rpm；200mL LB liquid is transferred in the ratio of 1:50 In culture medium, 28 DEG C, 200rpm, shaken cultivation 5 hours；5000rpm is centrifuged 15 minutes, collects thallus；It is resuspended in buffer In (5% sucrose, 0.03%Silwet L-77), OD is adjusted₆₀₀To 0.8.Inflorescence is inverted into 10 in mixed Agrobacterium bacterium solution Second, cover entire inflorescence with preservative film, remove preservative film after 24 hours, continue to be incubated at culturing room (22 DEG C, illumination in 14 hours/ 10 hours dark), until harvest seed (Clough and Bent, 1998).

The screening of 4 arabidopsis positive seedlings

(1) the arabidopsis thaliana transformation seed of harvest is impregnated 2 minutes in 70% ethyl alcohol, removes supernatant；

(2) liquor natrii hypochloritis impregnates further progress disinfection treatment in 5 minutes, with rinsed with sterile water 3-5 times；

(3) arabidopsis seed is sowed on the 1/2MS culture medium flat plate containing 50 μ g/mL antibiotic Kan；

(4) if on shuttle vector have Kan resistance reporter gene, by culture plate be placed in illumination cultivation room (22 DEG C, 16 Hour illumination/8 hour dark) culture 7-10 days；Selection is grown fine after Kan is screened, and true leaf blade and growing point are in deep Green and root can penetrate the plant of culture medium, primarily determine as positive seedling, during transplanting is buried.

5 real-time quantitative RT-PCRs

Using transgenic arabidopsis T2 generation and using each organ of wax plum as material, total serum IgE is extracted with Trizol kit, is then used The reverse transcription of PrimeScript RT reagent Kit (Takara, Japan) progress the first chain of cDNA.The first chain of cDNA is dilute PCR reaction is carried out as template after releasing 10 times.Then SYBR Premix Ex Taq (Bio-Rad, USA) reaction reagent is utilized Box completes reaction in Bio-Rad CFX96 fluorescence quantitative PCR instrument (Bio-Rad, the U.S.).It analyzes data obtained and counts Calculate target gene concentration in sample.PCR reaction system is 10 μ l, and 3 repetitions are arranged altogether, and reaction condition is 40 and recycles, 95 DEG C 60 seconds, 95 DEG C 5 seconds, 60 DEG C 34 seconds.

6 transgenic arabidopsis strain CpWOX13 and its endogenous gene expression analysis

(1) transgenic arabidopsis CpWOX13 gene expression amount detects

In order to further verify expression of the CpWOX13 gene in the arabidopsis of transgenosis, take wild type quasi- Southern mustard and transgenic arabidopsis extract total serum IgE compared with leaflet tablet 4-6 piece (about 400mg), with Trizol method, reverse transcription be cDNA as Template, internal reference select arabidopsis AtActin gene, carry out to the expression quantity of CpWOX13 gene in transgenic arabidopsis single plant real When fluorescence quantitative PCR detection (Fig. 3), 3 technologies are set and are repeated.The expression of extremely low CpWOX13 gene is detected in WT single plant (expression quantity 0.00003), CpWOX13 expression quantity is above wild type in 14 transgenosis single plants.14 in detection turn base In the arabidopsis single plant of cause, the expression of CpWOX13 gene differs greatly, wherein the expression quantity highest (expression quantity of strain 8# For 8.23386) be about WT 2.7 × 10⁵Times, the expression quantity of strain 1# minimum (expression quantity 0.72936) but also be WT 2.4 ×10⁴Times, illustrate that CpWOX13 expression quantity significantly raises in transgenosis single plant.Other transgenosis single plant expression quantity are from high to low 2#,7#,3#,6#,4#.Analysis quantitative result selects high expression quantity CpWOX13/8#, medium expression quantity CpWOX13/3# and low table Transgenic line up to amount CpWOX13/6# to indicate variable expression, carries on subsequent Phenotypic Observation and endogenous gene is fixed Amount analysis experiment.Wherein 2 times close to low expression amount CpWOX13/6# of high expression quantity CpWOX13/8#, significant difference.

(2) transgenic arabidopsis endogenous gene expression analysis

To study CpWOX13 gene concrete function, from the CpWOX13 gene pairs arabidopsis endogenous gene for being transferred to arabidopsis Influence is started with.It extracts transgenic arabidopsis strain respectively and wildtype Arabidopsis thaliana RNA, specific steps sees above, set according to sequence It counts special primer and carries out real-time fluorescence quantitative PCR detection (Fig. 4).The results show that being transferred to for CpWOX13 leads to all 10 quasi- south The expression quantity of mustard endogenous gene increases, and compared with wild type, the expression quantity of all genes is dramatically increased, minimum all increases 2 times, illustrate that bloom relevant gene of CpWOX13 gene pairs has facilitation.Wherein, become civilized integron AtSOC1 and AtFT table Reveal consistent trend, CpWOX13/3# expression quantity is higher probably due to the protein product of FT coding is from Ye Zhongzhuan in AtFT It is transported to and is expressed in SAM, and quantitative material is the fresh blade of arabidopsis, therefore AtFT trend is different from other floral genes.Research Show that WUS gene and AG gene form WUS/AG feedback regulation ring, quantitative result then shows CpWOX13 and AtAG expression quantity positive Close, i.e., AtAG expression quantity by CpWOX13 regulation, the latter can activate to a certain extent the former express.CpWOX13 gene is led Enter, have an impact to A, B, C genoid, AP1, PI and AG show up-regulation trend, illustrate the function and flower of CpWOX13 gene Growth course connection is close.The key gene that quantitative result can be seen that each flower induction approach has expression quantity increased Situation illustrates that the not single a certain item of participation of CpWOX13 is bloomed the regulation of approach, but is joined in the form of regulating and controlling key gene With a plurality of approach, converting at flower to Accelerate bloom for plant is adjusted.

7 transgenic arabidopsis phenotypic analyses

(1) advance flowering period: referring to the growing state of WT under same growth conditions, transgenic arabidopsis T is observed and recorded₂Generation The upgrowth situation of homozygous lines, counts and analysis is found, just each material time point (time of such as bolting, first of development The time that the flowers are in blossom, first fruit pod time of occurrence) for, transgenic plant is earlier than wild type (such as Fig. 5).

Observation as the result is shown (table 5), on time of bolting (bolting mark is set to bolting to 1cm or so), 35S:: CpWOX13/3#, 35S::CpWOX13/6# and 35S::CpWOX13/8# significant difference compared between WT, all earlier than WT, and three Difference is not then significant between person；Equally, compare first time that the flowers are in blossom, 35S::CpWOX13/3#, 35S::CpWOX13/6# With 35S::CpWOX13/8# also all earlier than WT and significant difference, and then no significant difference each other；In first fruit pod of knot Time upper three strains are with WT in the presence of there were significant differences, in addition, 35S::CpWOX13/8# strain is respectively at 35S:: CpWOX13/6# strain and 35S::CpWOX13/3# strain without significant difference, but after there is significant difference between the two；Transgenosis Three strains of arabidopsis be punished for being related to compared with wildtype Arabidopsis thaliana leaf quantity than the reduction of wild type (Fig. 5, table 5), difference Significantly；In addition, transgenic arabidopsis stem leaf general increase, high expression quantity strain and WT significant difference.In conclusion from wax plum From the point of view of CpWOX13 gene heterogenous expression arabidopsis phenotype, CpWOX13 gene may have following function:

1. promoting the generation of thaliana flower separate living tissue；

2. promoting the growth of plant, plant longitudinal growth may be promoted by way of extending plant life cycle；

3. it is related with stem leaf growth, i.e., it is related with the development of lateral organ.

5 turns of CpWOX13 gene arabidopsis strain index of correlation observations of table

Note: data mode in table are as follows: mean+SD；A, b, c, d, e, f indicate the horizontal upper significant difference of P < 0.05

(2) plant height is got higher:

Three strain plant heights compared with wildtype Arabidopsis thaliana of transgenic arabidopsis are all got higher, 35S::CpWOX13/3#, 35S::CpWOX13/8# plant height compared with WT increases about 12cm, 35S::CpWOX13/6# and the plant height increasing compared between WT 10cm is added, and there is significant difference (Fig. 5, table 5).

(3) root system is elongated, lateral root increases:

The growing state of the root of homozygous transgenic plant is observed, when finding the sprouting of transgenosis and nontransgenic plants root Between it is very nearly the same, but the speed of growth is slightly different, and wildtype Arabidopsis thaliana is obviously partially slow, while the seed sowed, the quasi- south of transgenosis Mustard root long is considerably longer than wildtype Arabidopsis thaliana (such as Fig. 6).Meanwhile in the comparison of growing way almost the same transgenic arabidopsis and WT Middle discovery, the lateral root branch on transgenic arabidopsis main root are significantly more than wild type (such as Fig. 6).Show WOX13 gene as weight The plant stem cell controlling gene wanted, CpWOX13 gene are transferred to the activity for being likely to affect arabidopsis Root apical meristem, Promote the differentiation of lateral root.

CpWOX13 gene may have the function of promotion plant early blossoming, and CpWOX13 can raise other early blossoming bases in other words Cause achievees the purpose that promote flowering of plant and growth；WUS gene is natively important plant stem cell controlling gene, and WOX13 gene is even more the growth course for directly participating in root, therefore the heterogenous expression of CpWOX13 is likely to affect the arabidopsis tip of a root The activity of separate living tissue；In addition, the heterogenous expression meeting render transgenic Arabidopsis plant of CpWOX13 is got higher, it may for plant The raising of upper part biological amount has certain effect.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Sequence table

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Claims

1. wax plum CpWOX13 albumen, are as follows: the protein that the amino acid shown in SEQ ID No. 2 forms.

2. encoding the gene of albumen described in claim 1.

3. gene as claimed in claim 2, which is characterized in that its nucleotide sequence is as shown in SEQ ID No.1.

4. the carrier containing gene described in Claims 2 or 3.

5. the engineering bacteria containing gene described in Claims 2 or 3.

6. gene described in Claims 2 or 3 is at the regulation plant florescence and improves the application in biomass.

7. application as claimed in claim 6, which is characterized in that the plant is arabidopsis.

8. application of the gene described in Claims 2 or 3 in prepare transgenosis plant.

9. a kind of construction method of transgenic plant, which is characterized in that using the method for mediated by agriculture bacillus, by claim 4 institute The carrier stated is transferred in plant tissue, screening transgenic plant.