CN105039397A - Method for improving quality of cotton fibers by overexpressing GhCAD6 gene - Google Patents

Abstract

The invention relates to a method for improving quality of cotton fibers by overexpressing GhCAD6 gene, belonging to the technical field of biology. The method comprises the following steps: constructing a plant overexpression vector pCAMINBIA-2300-GhCAD6 from styrone dehydrogenase gene GhCAD6 in cotton fibers, transforming the vector into agrobacterium LB4404 by agrobacterium mediation, and integrating into cotton to obtain the trans-styrone dehydrogenase gene cotton plant. The field and molecular detection analysis detects that the lignin content in cotton fibers obviously increases in different development periods, the fibrocyte wall thickness obviously decreases in the cotton secondary wall rapid deposition stage, and the ripe cotton fiber surface becomes more compact and smooth. The method can maximally enhance the length of the cotton fibers by 16.935%, enhance the specific strength by 25.10% and enhance the fiber yield by 3.80%, thereby greatly improving the quality of the cotton fibers.

Description

By the method for overexpression GhCAD6 improvement of genes cotton fiber quality

One, technical field

The present invention relates to a kind of transgenic breeding method, is specifically a kind of method being improved cotton fibre quality by overexpression cinnamyl-alcohol dehydrogenase gene GhCAD6.

Two, background technology

Cotton is one of major economic crops of China, and China is also big producing country and the consumption big country of cotton in the world; Cotton fibre is the main raw material of people's clothing and daily textiles, in national economy and people's lives, occupy critical role.But Cotton in China fibrous quality is also unsatisfactory, be difficult to the multiple demand meeting domestic and international market and textile industry.Fibrous quality mainly deposits defect both ways: one is that fiber strength is on the low side, causes certain influence to the quality product of cotton yarn; Two is that the collocation of fibrous quality all indexs is unreasonable.After the mid-90 in 20th century, Cotton in China quality-improving enters the bottleneck phase, specific tenacity and other physical performance index do not match, although target is still emphasis while other quality parameter of maintenance improve fibre strength, but owing to lacking excellent germplasm system and parent material, also lack innovative technology, thus main quality index take on a new look and not obvious.

Along with the research of cell elongation correlation function gene, the approach of cotton fibre quality improvement turns to conventional breeding to combine with molecular breeding from conventional breeding.Although the transgene improvement of cotton fiber quality obtains some progress at present, but existing transgenosis is mainly for the improvement of cotton fiber length, be the foreign gene utilizing some directly related with elongate fiber, such as spider silk, elongation albumen etc. are improved cotton fiber.And the expression of most of foreign gene in cotton fiber can not genetic stability well, so the using value in Cotton Production is very limited.

Cinnamyl-alcohol dehydrogenase (CAD) is one and deposits a relevant important gene family with cell walls, grows in the growth of initial and secondary wall play an important role at secondary wall.This seminar early-stage Study each gene to cotton fiber CAD gene family has carried out expression analysis, find that the expression amount of GhCAD6 gene in cotton fiber is compared with higher in other tissue, and grow synchronous with the secondary wall of cotton fiber, during secondary wall deposition, expression amount is the highest.And find that the gene GhCAD6 of coding cinnamyl-alcohol dehydrogenase reaches predominant expression at Fibre Development middle and later periods (cotton secondary wall thickens the phase) expression amount, show that GhCAD6 gene may have important function in cotton fiber secondary wall formation process.

Therefore, we screen the candidate gene that GhCAD6 improves as fibrous quality, expect the quality being improved cotton fiber by regulation and control GhCAD6 gene at the expression level of cotton fiber.

But how to build desirable cinnamyl-alcohol dehydrogenase gene GhCAD6 expression vector, and determine pcr amplification primer and reaction system, and foundation is the key that cinnamyl-alcohol dehydrogenase gene GhCAD6 is able to practical application in cotton fiber quality improvement transgenic breeding to the evaluation and screening standards and measures of progeny material.Not yet there is relevant research and apply report at present.

Three, summary of the invention

The present invention aims to provide a kind of method by regulating the expression of gene in cotton fiber to carry out improving cotton fiber quality.Exactly be to provide the application method of cinnamyl-alcohol dehydrogenase gene (GhCAD6) in cotton fiber quality improvement breeding, the construction process of this expression vector and pcr amplification primer and reaction system are particularly provided, and set up the evaluation and screening standards and measures to progeny material.

The method being improved cotton fibre quality by overexpression cinnamyl-alcohol dehydrogenase gene GhCAD6 provided by the invention, GhCAD6 is built a plant overexpression carrier pCAMINBIA-2300-GhCAD6, by agriculture bacillus mediated method by this gene integration in cotton, obtain transfer-gen plant, plant through continuous multi-generation.By carrying out the comprehensive identification method of Molecular Detection, field test, electron-microscope scanning, fiber quality traits detection to progeny material, by for evaluation and screening, continuous 3 generation the transgenic progeny that is no longer separated of above genetic stability can be defined as effectively importing and improveing successful transgenic line, thus obtain staple length and strength raising, soluble sugar content significantly reduced cotton transgenic genetic stability progeny material.

Amplimer is the partial sequence zone design primer selecting intron in cross-domain genomic dna, and make the product of amplification only come from the encoding sequence of the GhCAD6 in transgene carrier, this primer is:

F primer 5 '-TGTGCAGGGGTGACAGTTTAC-3 ';

R primer 5 '-CCCAATAAAACTCCCTGTAATCG-3 ';

And its amplified reaction program is: after 94 DEG C of denaturation 5min, 94 DEG C of sex change 45s, 58 DEG C of annealing 45s, 72 DEG C extend 1min, carry out 35 circulations, and 72 DEG C extend 10min, 4 DEG C of preservations.

Described comprehensive identification method is

In T1-T2 generation, meets following 2 aspects simultaneously:

(1) kantlex is qualified: grow after 2-3 sheet true leaf until cotton, three times are screened respectively with 0.5%, 0.75%, 1% kantlex solution, bolting house is divided into 5-7 days, select the plant wiring mark with kalamycin resistance, gather blade, be placed in liquid nitrogen, detect for follow-up PCR;

(2) PCR detects positive: from the blade that above-mentioned steps is preserved, extract DNA, the primer described in claim 2 and response procedures is utilized to increase, PCR primer is put electrophoresis in 1.5% agarose, by ultraviolet gel imaging instrument observations, occur object band;

T3 generation and the later offspring that is not separated detect and meet:

(1) PCR detects positive: from the blade that above-mentioned steps is preserved, extract DNA, the primer described in claim 2 and response procedures is utilized to increase, PCR primer is put electrophoresis in 1.5% agarose, by ultraviolet gel imaging instrument observations, occur object band;

(2) fragment utilizing claim 2 primer to increase is the offspring that Southern hybridization check confirms as single copy insertion;

(3) detect that GhCAD6 gene raises at secondary wall thickening developmental stage expression amount by RT-PCR method;

(4) adopt Klason method to determine content of lignin in transgenic progeny material fiber to increase;

(5) the transmission electron microscope observation ripe transgenic progeny fiber finer cell wall degree of packing increases, and sem observation fiber surface polishes;

(6) specific tenacity of transgenic progeny material increases by more than 15%, and staple length increases by more than 10%, and mic value, ginning outturn and economical character index can both keep the proterties of original kind.

The present invention starts with from the Structure and Properties changing cotton fiber cell wall, phase predominant expression can be thickened at cotton fiber secondary wall according to cinnamyl-alcohol dehydrogenase gene (GhCAD6), and participate in synthesis, the deposition of the synthesis of xylogen in cotton fiber and fiber finer cell wall.By building cinnamyl-alcohol dehydrogenase gene (GhCAD6) Overexpression vector, build high specific pcr amplification primer and reaction system, successfully realize goal gene to import, and by setting up transgenic progeny standard of perfection and screening method, obtain the progeny material of genetic stability, realize the practical application of cinnamyl-alcohol dehydrogenase gene (GhCAD6).Its target gene select correct, expression vector establishment and design of primers rationally, offspring's standard of perfection and method strict, by force comprehensive, ensure that improved effect.

The method achieve GhCAD6 gene overexpression in cotton fiber, and prove that the strain through improvement can genetic stability, thus reach the object of improving cotton fiber quality, for the combination promoting Molecular breeding in upland cotton: and conventional breeding, there is positive effect.

Four, accompanying drawing explanation

Fig. 1 is that the GhCAD6 gene that E6 starts is inserted into the structure schematic diagram of the overexpression carrier E6::GhCAD6 obtained in carrier pCAMBIA-2300.Wherein LB and RB represents the right boundary sequence of T-DNA respectively; NPTII represents kanamycin gene; E6 represents cotton fiber specific E6 promotor; GhCAD6 represents goal gene; LacZ represents beta-galactosidase gene; NospolyA represents Opines synthase terminator.

Fig. 2 is E6::GhCAD6 offspring and contrast PCR detection figure, and in figure, M represents DL2000Marker; " new land early 36 " strain that 1-5 represents that E6::GhCAD6 turns respectively, 6 represent blanks, and 7 represent not genetically modified acceptor material " new land early 36 ", and 8 represent the overexpression vector plasmid built.

Fig. 3 is that E6::GhCAD6 offspring and contrast " new land early 36 " T6Southern hybridizes figure.In figure, CK-represents not genetically modified acceptor material " new land early 36 "; CK+ represents the overexpression vector plasmid of structure; 1,2,3,4 represent that E6::GhCAD6 turn " new land early 36 " T6 strain.

Fig. 4 be GhCAD6 gene at cotton fibre different development stage gene expression analysis figure, 1 represents GhUBI gene, 2-6 represents that different GhCAD6 turns " new land early 36 " T6 strain gene.

Fig. 5 adopts Klason method to measure cotton fiber content of lignin change microscopic comparison for turning E6::GhCAD6 offspring and contrast " new land early 36 ", wherein a, c, e, g, i be respectively contrast cotton fiber development 20,25,30,40DPA and the content of lignin in stage of maturity; B, d, f, h, j be respectively transgenic progeny cotton fiber development 20,25,30,40DPA and the content of lignin in stage of maturity.

Fig. 6 is for turning E6::GhCAD6 offspring and contrast " new land early 36 " cotton fiber cell wall thickness change microscopic comparison.Wherein a, c, e, g, i be respectively contrast cotton fiber development 20,25,30,40DPA and the cell walls form in stage of maturity; B, d, f, h, j be respectively transgenic progeny cotton fiber development 20,25,30,40DPA and the cell walls form in stage of maturity.

Fig. 7 is for turning E6::GhCAD6 offspring and contrast " new land early 36 " cotton fiber cell wall thickness change curve.

Fig. 8 is for turning E6::GhCAD6 offspring and contrast " new land early 36 " cotton mature fibers microscopic comparison.

Five, embodiment

The step used in following embodiment and method if no special instructions, are Molecular Detection field routine and generic case, the understanding that term and abbreviation are conducive to technical solution of the present invention.

Material used in following embodiment, reagent etc. if no special instructions, are all that business buys acquisition.

Be GhCAD6 gene is proceeded to Upland Cotton " new land early 36 " in following embodiment, progeny material is for turning E6::GhCAD6 offspring and contrast " new land early 36 " offspring's (wild-type).

(1) GhCAD6 overexpression vector construction

1. plasmid extraction

Respectively by carrying GhCAD6cDNA sequence carrier T, the E.coliDH5 α 40 μ L bacterium liquid of E6-pCAMBIA-2300 carrier is placed in the LB liquid nutrient medium that 4mL contains 100mg/L penbritin, 37 DEG C, 200rpm incubated overnight, extracts plasmid;

2. the enzyme of carrier and goal gene is cut

Double digestion system: ddH2O20.5 μ L, plasmid (E6-pCAMBIA-2300 and GhCAD6cDNAT carrier) 20 μ L, 10 × Buffer damping fluid 5 μ L, BSA0.5 μ L, restriction enzyme SacI2 μ L, restriction enzyme BamHI2 μ L, cumulative volume 50 μ L.

3. the recovery of digestion products

Digestion products reclaims, and uses the universal DNA purifying in sky root biochemistry (Beijing) to reclaim test kit.

4. carrier is connected with goal gene, transforms and identifies

(1) connect

The digestion products of recovery to be spent the night connection according to following system 16 DEG C:

Linked system: ddH2O5.5 μ L, E6-pCAMBIA-2300 carrier 1 μ L, GhCAD6 fragment 2 μ L, 10 × Buffer damping fluid 1 μ L, T4 ligase enzyme 0.5 μ L, cumulative volume 10 μ L.

(2) product conversion E.coliDH5 α is connected

Product conversion DH5 α competent cell will be connected, carry out bacterium liquid PCR to after conversion bacterium colony shaking culture.Reaction system is as follows: 10 × PCRMix1 μ L, ddH2O6.4 μ L, bacterium liquid 1 μ L, F primer 0.5 μ L, R primer 0.5 μ LdNTP0.4 μ L, Taq DNA polymerase 0.2 μ L, cumulative volume 10 μ L.

The primer sequence used is: F primer 5 '-AAGTTGGGTCAGATGTAAGC-3 '

R primer 5 '-TAAGCATTTCCTCTGTTTCC-3 '

PCR response procedures: after 94 DEG C of denaturation 5min; 94 DEG C of sex change 45s, 58 DEG C of annealing 45s, 72 DEG C extend 1min, carry out 35 circulations; 72 DEG C extend 10min, 4 DEG C of preservations.

By above-mentioned PCR primer, after containing 1% agarose gel electrophoresis of dyestuff, observe.

(3) enzyme cuts qualification

Select PCR positive colony, extract plasmid, carry out enzyme and cut qualification.It is as follows that enzyme cuts system:

Double digestion system: ddH2O2.35 μ L, plasmid (E6-pCAMBIA-2300-GhCAD6) 5.0 μ L, 10 × Buffer damping fluid 1.5 μ L, BSA0.15 μ L, restriction enzyme SacI0.5 μ L, restriction enzyme BamHI0.5 μ L, cumulative volume 10 μ L.

Select enzyme to cut the desirable bacterium liquid of result, add equal-volume 30% glycerine, protect bacterium for-80 DEG C.

Fig. 1 is shown in by the plant overexpression carrier structure schematic diagram built.

In Fig. 1, LB and RB represents the right boundary sequence of T-DNA respectively; NPTII represents kanamycin gene; E6 represents cotton fiber specific E6 promotor; GhCAD6 represents goal gene; LacZ represents beta-galactosidase gene; NospolyA represents Opines synthase terminator.

(2) Agrobacterium-medialed transformation

1. prepare Agrobacterium LBA4404 competence and use freeze-thaw method to transform

2. the detection of Agrobacterium LBA4404 recon

The single bacterium colony of Agrobacterium on picking transformation plate, be inoculated in liquid YEB substratum (containing 25mg/L Rifampin, 50mg/L kantlex) in 28 DEG C, 200rpm shaking culture spends the night, carry out bacterium liquid PCR and detect expression vector and whether proceeded in Agrobacterium LBA4404.The reaction system that bacterium liquid detects, primer and response procedures connect product conversion E.coliDH5 α in (one)-4-(2) method with is above consistent.

Select PCR to detect positive clone to carry out preserving and follow-up conversion.

3. Agrobacterium uses the flower-dipping method converting cotton of improvement

1mL bacterium liquid is placed in the 1L substratum containing (containing 50mg/L Rifampin, 50mg/L kantlex), 28 DEG C are cultured to bacterium liquid OD600 and are greater than more than 1.2;

8000rpm4 DEG C of centrifugal 10min collects bacterium liquid, bacterium liquid is resuspended in 10% sucrose solution that 1L contains 0.2%Silwet-L77, carries out transformation experiment in field;

Same day flower is selected to burst forth completely, but the cotton flower of also non-Pollination Fertilization, with small-sized watering can facing to cotton column cap spray 2-3 time (about 1 ~ 2mL bacterium liquid); Closed up by flower petal, tie with small-sized rubber case, the colored knitting wool of anthocaulus is pinioned, and as mark, transforms and terminates rear timely irrigation.

Treat the harvest season, transform flower according to label collection, cotton ginning, lint, obtain T0 seed.

(3) offspring's plantation and evaluation and screening

By the plantation of the T0 seed of results, grow after 2 ~ 3 true leaves until cotton, screen three times with 0.5%, 0.75%, 1% kantlex solution respectively, bolting house is divided into 5 ~ 7 days.Select the plant (dripping kantlex position not turn yellow) with kalamycin resistance, wiring marks, and gathers blade, is placed in liquid nitrogen, detects for follow-up PCR.

Extract test kit (Tiangen) operation instruction by plant genome DNA, be stored in from abovementioned steps in the fresh tender leaf in liquid nitrogen and extract DNA.The reaction system that PCR detects and response procedures connect product conversion E.coliDH5 α in (one)-4-(2) method with is above consistent, and amplified fragments size is 501bp.

The primer sequence used is: F primer 5 '-TGTGCAGGGGTGACAGTTTAC-3 '

R primer 5 '-CCCAATAAAACTCCCTGTAATCG-3 '

PCR primer is placed in 1.5% agarose electrophoresis, by ultraviolet gel imaging instrument (Bio-Rad) observations, occurs that the preliminary screening of object band is transgenic progeny.

E6::GhCAD6 offspring and contrast PCR detection figure are shown in Fig. 2.

Treat the harvest season, select the arable material of cotton plant plant type, results.Roll taking cotton fiber, send the Ministry of Agriculture (Xinjiang) cotton quality supervision and inspection center to carry out fiber quality traits detection.

Retain the transgenic progeny strain that PCR detects the positive, staple length significantly increases, as the material in next one plantation season, other materials is as Germ-plasma resources protection.

Continuous 3 generation the transgenic progeny that is no longer separated of genetic stability can be defined as effectively importing and improveing successful transgenic line.

(4) Southernblotting testing goal gene copy number

1. cotton genomic dna extracts

Select the acceptor material of each five strains of transgenic progeny of two kinds of overexpression carriers.Gather tender leaf, extract genomic dna according to the following step.

Get fresh young leaflet tablet about 2g, defoliation arteries and veins, add liquefied ammonia grinding (three times) in Powdered.

Add 3mL cell pyrolysis liquid, proceed in the 7.0mL centrifuge tube of precooling, put on ice.After sample all processes, thermal agitation mixes.

4 DEG C, 12000rpm, centrifugal 10min; Abandon supernatant, add 3mLDNA extracting solution (65 DEG C of preheatings in advance), mixing of fully vibrating, 65 DEG C of water-bath 30 ~ 45min, slowly put upside down 3 ~ 4 times therebetween, take out after 40min, 4 DEG C of cooling 10min.

Add isopyknic chloroform/primary isoamyl alcohol (V/V=24: 1).4 DEG C, 12000rpm, centrifugal 15min.

Supernatant is moved in new 7mL centrifuge tube, add the pre-cold isopropanol of 0.8 times of volume, slowly mix, place more than 30min for-20 DEG C.

4 DEG C, 12000rpm, centrifugal 15min, abandons supernatant, removes raffinate.Add 1.5mL70% ethanol, washing precipitation.Repeated washing once.

4 DEG C, 12000rpm, centrifugal 10min, abandons supernatant, adds 500 μ LTE dissolution precipitation 1h.

Add isopyknic chloroform/primary isoamyl alcohol, extracting; 4 DEG C, 12000rpm, centrifugal 10min.

Supernatant is moved in new EP pipe, add the dehydrated alcohol of 2 times, after mixing, leave standstill 1h.

4 DEG C, 12000rpm, centrifugal 10min, abandons supernatant, dries up precipitation, 50 μ LddH2O dissolution precipitations, measures concentration ,-20 DEG C of preservations.

Southernblotting uses Roche digoxigenin labeled test kit I.

2. goal gene pcr amplification

Use following system for pcr amplification.

Reaction system is as follows: 10 × PCRMix1 μ L, ddH2O37.5 μ L, object fragment 1 μ L, 10 × Buffer damping fluid 5.0 μ L, dNTP4 μ L, F primer 1.0 μ L, R primer 1.0 μ L, dNTP0.4 μ L, LA-TaqDNA polysaccharase 0.2 μ L, cumulative volume 50 μ L.

The primer sequence used is: F primer: 5 '-TGTGCAGGGGTGACAGTTTAC-3 '

R primer: 5 '-CCCAATAAAACTCCCTGTAATCG-3 '

Plasmid is the carrier with goal gene.

PCR response procedures: after 94 DEG C of denaturation 5min, 94 DEG C of sex change 45s, 58 DEG C of annealing 45s, 72 DEG C extend 1min, carry out 35 circulations, and 72 DEG C extend 10min, 4 DEG C of preservations.

PCR primer reclaims, and uses the universal DNA purifying in sky root biochemistry (Beijing) to reclaim test kit.

3. the mark of probe (PCR primer)

1 μ gPCR product is added aseptic deionized water to 16 μ L, mixing;

Sex change PCR primer, ice bath cooling immediately after boiling water bath 10min;

Add the Dig-Highprime of 4 μ L, brief centrifugation, hatch 20h for 37 DEG C; 65 DEG C of process 10min ,-20 DEG C of preservations.

4. plasmid and genomic dna enzyme are cut

Get each 20 μ g of DNA, use BamHI enzyme to cut through night, it is as follows that enzyme cuts system:

DdH2O23.0 μ L, DNA20.0 μ L, 10 × Buffer damping fluid 5.0 μ L, BSA0.15 μ L, restriction enzyme XbaI2 μ L, cumulative volume 50 μ L.

Electrophoresis detection enzyme cuts effect, cuts effect according to enzyme, carries out next step operation.

5. electrophoresis and transferring film

Whole digestion products is added in 0.7% sepharose, electrophoresis.Electrophoresis process is after 120V electrophoresis 1h, during with 30V electrophoresis to tetrabromophenol sulfonphthalein distance gel edges about 1.5em, terminates electrophoresis.

Use neutral sex change liquid sex change 30min.

Neutralizer neutralization twice, first time in and 30min, second time in and 15min; Period cuts the positively charged nylon membrane of a size large 0.5cm more each than gel four limit, and nylon membrane distilled water infiltrates and is placed on 5min in transfering buffering liquid.

Neutralization terminates, according to the operation assembling transfer device of Molecular Cloning: A Laboratory guide (second edition).Transfering buffering liquid is 20 × SSC, transfer 12h.

Transferring film terminates, and 2 × SSC washs, 120 DEG C of fixing 30min.

6. hybridization and detection

Illustrate according to test kit and prepare hybridization solution.The hybridization solution of certain volume is added in hybridization bucket, then film is transferred in hybridization bucket, be placed in hybrid heater 42 DEG C of prehybridization 30min.

By the probe boiling water bath 5min marked, ice bath at once.The probe of sex change is joined in hybridization solution according to 1: 2500.

Prehybridization terminates, and pours out hybridization solution, adds the hybridization solution containing probe of certain volume.42 DEG C of hybridized overnight.

Hybridization terminates, and under room temperature, 2 × SSC, 0.1%SDS wash 5min; At 65 DEG C, 0.5 × SSC, 0.1%SDS wash 15min.

Lavation buffer solution washing 5min, repeats 2 times.

100mL confining liquid closes 30min.

50mL antibody-solutions 30min.

200mL lavation buffer solution washing 15min, repeats 2 times.

100mL detects damping fluid balance 5min.

50mL nitrite ion lucifuge develop the color, until color spot occur and clear.DdH2O washs termination reaction, takes a picture.

Overexpression transgenic line Southernblotting the results are shown in Figure 3.

In Fig. 3, CK-represents not genetically modified acceptor material; CK+ represents the overexpression vector plasmid of structure; 1,2,3,4 represent that E6::GhCAD6 turn " new land early 36 " T6 strain.

The Southernblotting result of control material and transgenic progeny material is compared analysis, finds that GhCAD6 gene exists single copy in progeny material.

(5) GhCAD6 gene is at cotton fibre different development stage gene expression analysis

To the cotton fibre sample total serum IgE reverse transcription of extracted different strain different development stages, then carry out Semiquatitative RT-PCR assay expression analysis.Result shows in transgenic line and contrast, and the expression amount of GhCAD6 increases along with the increase in cotton fiber development period.Before 15DPA, the destination gene expression amount of transgenic progeny and contrast is all very low, and when 20DPA, GhCAD6 expression amount starts remarkable increase.When 20DPA and 25DPA, the expression amount of transgenic progeny goal gene is all higher than contrast.Illustrate that the expression time of GhCAD6 in transgenic progeny strain will be grown, expression amount is higher, has a certain impact to cotton fiber quality tool.

Fig. 4 is that GhCAD6 gene is at cotton fibre different development stage gene expression analysis figure.Wherein 1 is GhEF1 α; 2-5 is that E6::GhCAD6 turns " new land early 36 " T6 strain; 6 is contrast.Result display in figure: the expression amount of GhCAD6 increases along with the increase in cotton fiber development period.Before 15DPA, the destination gene expression amount of transgenic progeny and contrast is all very low, and when 20DPA, GhCAD6 expression amount starts remarkable increase.When 20DPA and 25DPA, the expression amount of transgenic progeny goal gene is all higher than contrast.

(6) transgenic progeny fibrous quality measures

To the continuous three generations of the offspring turning E6::GhCAD6 gene (T4-T6) fibrous quality measurement result with compare, find that the fiber quality characteristics of transgenic progeny strain generally improves, genetic stability rear 3 generation staple length and strength rate of increase in table 1, the different index of T6 material fiber quality with contrast relatively in table 2.

Table 1 turn E6::GhCAD6 offspring continuous 3 generation fibrous quality

Table 2 turns E6::GhCAD6T ₆fiber quality traits compares

(7) Klason method measures content of lignin in transgenic progeny fiber

1. the pre-treatment of cotton fiber

Take out the cotton boll that shells deposited early stage from-20 DEG C of refrigerators, be placed in homogenization buffered soln and soak, with tweezers, cotton fibre is separated with cottonseed, notice that the fiber be separated can not with impurity such as seed coats.The cotton fibre of separation is immersed in the beaker that homogenization buffered soln is housed, avoids producing gossypol browning, and clean immediately.After taking off seed for the ripe cotton fibre of dehydration with small-sized cotton gin, recycled fiber is for subsequent use.

2. the cleaning of cotton fibre

Cotton fibre homogenization buffer solution for cleaning twice, 80% acetone washes twice, and pure acetone is washed once, all uses metal clip garlic device to be extracted by the solution in fiber at every turn.Afterwards washed cotton fibre is placed on filter paper, 45 DEG C of dry for standby.

3. the mensuration of content of lignin

Accurately take 2g (being accurate to 0.001) sample, add 15mL72% sulfuric acid, ambient temperatare puts 2h makes cotton fibre degradable; Reaction solution being diluted with water to sulfuric acid concentration is 3%, 0.1MPa, 121 DEG C, digestion 1h; Taking-up cools 20min under being placed in room temperature, the filter paper filtering solution dried to constant weight with 60 DEG C, and with distilled water flushing filter paper until it is neutral; Filter paper is put into baking oven together with residue, 60 DEG C, dry to constant weight and obtain xylogen.

Content of lignin (%)=(W2-W1-W3)/2.00 × 100%

In formula: W ₁the weight (g) of-filter paper; W ₂-dry the weight (g) of rear filter paper together with residue; W ₃-ash content heavy (g).

To turning E6::GhCAD6 progeny T ₆in generation, compares in table 3 with control material content of lignin.Result shows, transgenic line improves a lot than contrast content of the same period at the content of lignin of different development stage.

The content of lignin of table 3 transgenic progeny and control group

Fig. 5 for turning E6::GhCAD6 offspring and contrast adopts Klason method to measure cotton fiber content of lignin change microscopic comparison, wherein a, c, e, g, i be respectively contrast cotton fiber development 20,25,30,40DPA and the content of lignin in stage of maturity; B, d, f, h, j be respectively transgenic progeny cotton fiber development 20,25,30,40DPA and the content of lignin in stage of maturity.Result display in figure: transgenic line improves a lot than contrast content of the same period at the content of lignin of different development stage.

(8) cotton fiber cell wall deposition conditions is analyzed and mature fibers observation

1. TEM sample preparation and observation

(1) fixing: immediately cotton fibre sample to be thrown in the medical vials that 4% glutaraldehyde (phosphoric acid buffer of 0.1mol/L, pH7.2) is housed after sampling and first fix, after be put into 4 DEG C of Refrigerator stores to whole sample and take;

(2) embed in advance: cotton fibre is embedded in advance, take out stretching on filter paper by cotton fibre from 4% glutaraldehyde, cut middle part with blade and be about 0.3cm, be put in embedding plate with tweezers and straighten, embed in advance in embedding plate with 3.5% (agar powder and the configuration of 0.1mol/L phosphoric acid buffer) agar, solidify the part that rear excision is unnecessary, the fiber of only remaining embedding, then throw in medical vials;

(3) rinse: rinse sample 3h with 0.1mol/L phosphoric acid buffer;

(4) fix again: 1% osmic acid (identical damping fluid) fixes 2h again;

(5) rinse again: then rinse 1h with 0.1mol/L phosphoric acid buffer again;

(6) dewater: be that 30,50,70,80,90,95,100% alcohol dewaters step by step through concentration respectively, every grade of dehydration 20min, wherein 100% need dewater 1h;

(7) replace: use 1: 1 (alcohol/acetone) to soak 1h again, 1: 4 (alcohol/acetone) soaks 80min, pure acetone leaching 1h, 1: 1 (epoxy resin/acetone) soaks 2h, 3: 1 (epoxy resin/acetone) soak 3h, 5: 1 (epoxy resin/acetone) soak 4d, and pure epoxy resin (Epon812) soaks 1d and replaces;

(8) embed: with pure epoxy resin embedded material in embedding plate, be put in baking oven and at 37,45,60 DEG C, respectively place 1d respectively;

(9) cut into slices: with German LEICAULTRACUTR microtome;

(10) dyeing and observing is taken pictures: 2% uranyl acetate and 6% lead citrate dye each 20min, distilled water i.e. observable after rinsing and drying; Take pictures with FDAC H-600 transmission electron microscope observing.

2. measure cell secondary wall thickness

Get the transmission electron micrograph of 10DPA, 15DPA, 20DPA, 25DPA, 30DPA and the observation of ripe cotton fiber.Cell secondary wall thickness is measured with AdobePhotoshop image processing software.Get 15 figure each period at random, each figure is uniformly distributed and gets 15 point measurement secondary wall vertical thickness and average.

FT(μm)＝[(x ₂-x ₁) ²+(y ₂-y ₁) ²)] ^1/2/DPμm

In formula, FT (μm) represents fiber finer cell wall secondary wall thickness, (x1, y1) is for enclosing certain point coordinate, (x2 in secondary wall thickening, the coordinate of the peripheral vertical line point of secondary wall y2) corresponding to certain point, DPm is the pixel count of every micron.

Data acquisition Excel software carries out statistical study.

Fig. 6 carries out transmission electron microscope observing and the result of taking pictures to transgenic line T6 and control material, wherein a, c, e, g, i, k be respectively contrast cotton fiber development 10,15,20,25,30DPA and the cell walls form in stage of maturity; B, d, f, h, j, l be respectively transgenic progeny cotton fiber development 10,15,20,25,30DPA and the cell walls form in stage of maturity; PCW is primary wall; SCW is secondary wall.

Cotton fiber cell wall deposition conditions analysis in Fig. 6 and mature fibers observations show, transgenic line at the 10DPA-15DPA cell wall thickness being and the thickness no significant difference contrasted, from 20DPA to mature period cell walls than contrast of the same period consolidation more.

Fig. 7 is that the cell secondary wall thickness profiles figure obtained measured by above-mentioned transmission electron microscope picture through image processing software.

3. SEM sample preparation and observation

Respectively the ripe cotton fiber of transgenic line and control material is put into 95% ethanol: after ether (1: 1) degreasing 10min, then the galley proof product after degreasing are placed in 100% ethanol clean 2min, taking-up filter paper blots or seasoning.With IB-5 type ion sputtering instrument spraying plating platinum film, thickness 10nm.Under SUPRA55VP type scanning electron microscope, observe cotton fiber form and take pictures.

To transgenic line T ₆and control material carry out above-mentioned electron-microscope scanning after observe, result shows, the ripe cotton fiber surface of transgenic line is more smooth than the fiber of contrast to compact, and thinner.Scanning electron microscope comparing result is shown in Fig. 8.In Fig. 8, a is contrast cotton mature fibers electron-microscope scanning figure; B is transgenic progeny cotton mature fibers electron-microscope scanning figure.

Claims (3)

1. the method for cotton fibre quality is improved by overexpression cinnamyl-alcohol dehydrogenase gene GhCAD6, GhCAD6 is built a plant overexpression carrier pCAMINBIA-2300-GhCAD6, by agriculture bacillus mediated method by this gene integration in cotton, obtain transfer-gen plant, through planting 3-6 generation continuously, by carrying out Molecular Detection to progeny material, field test, electron-microscope scanning, the comprehensive identification method that fiber quality traits detects, by for evaluation and screening, continuous 3 generation the transgenic progeny that is no longer separated of above genetic stability can be defined as effectively importing and improveing successful transgenic line, thus obtain staple length and strength raising, soluble sugar content significantly reduced cotton transgenic genetic stability progeny material.

2. the method for improvement cotton fibre quality according to claim 1, it is characterized in that described overexpression carrier pCAMINBIA-2300-GhCAD6 transforms amplimer is the partial sequence zone design selecting intron in cross-domain genomic dna, is specially:

F primer 5 '-TGTGCAGGGGTGACAGTTTAC-3 ';

R primer 5 '-CCCAATAAAACTCCCTGTAATCG-3 ';

And its amplified reaction program is: after 94 DEG C of denaturation 5min, 94 DEG C of sex change 45s, 58 DEG C of annealing 45s, 72 DEG C extend 1min, carry out 35 circulations, and 72 DEG C extend 10min, 4 DEG C of preservations.

3. the method for improvement cotton fibre quality according to claim 1 and 2, is characterized in that described comprehensive identification method is

In T1-T2 generation, meets following 2 aspects simultaneously:

(1) kantlex is qualified: grow after 2-3 sheet true leaf until cotton, three times are screened respectively with 0.5%, 0.75%, 1% kantlex solution, bolting house is divided into 5-7 days, select the plant wiring mark with kalamycin resistance, gather blade, be placed in liquid nitrogen, detect for follow-up PCR;

(2) PCR detects positive: from the blade that above-mentioned steps is preserved, extract DNA, the primer described in claim 2 and response procedures is utilized to increase, PCR primer is put electrophoresis in 1.5% agarose, by ultraviolet gel imaging instrument observations, occur object band;

T3 generation and the later offspring that is not separated detect and meet:

(1) PCR detects positive: from the blade that above-mentioned steps is preserved, extract DNA, the primer described in claim 2 and response procedures is utilized to increase, PCR primer is put electrophoresis in 1.5% agarose, by ultraviolet gel imaging instrument observations, occur object band;

(2) fragment utilizing claim 2 primer to increase is the offspring that Southern hybridization check confirms as single copy insertion;

(3) detect that GhCAD6 gene raises at secondary wall thickening developmental stage expression amount by RT-PCR method;

(4) adopt Klason method to determine content of lignin in transgenic progeny material fiber to increase;

(5) the transmission electron microscope observation ripe transgenic progeny fiber finer cell wall degree of packing increases, and sem observation fiber surface polishes;

(6) specific tenacity of transgenic progeny material increases by more than 15%, and staple length increases by more than 10%, and mic value, ginning outturn and economical character index can both keep the proterties of original kind.