CN109295029A

CN109295029A - Application of the cotton class calcineurin B subunit gene GhCBL1 in cotton and tobacco breeding

Info

Publication number: CN109295029A
Application number: CN201811352984.4A
Authority: CN
Inventors: 阎星颖; 白文钦; 汪鸿星; 曾健晏; 侯磊; 裴炎
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2018-11-14
Filing date: 2018-11-14
Publication date: 2019-02-01

Abstract

The invention belongs to field of plant genetic project technology, and in particular to application of the cotton class calcineurin B subunit gene GhCBL1 in cotton and tobacco breeding.By constructing the overexpression carrier of the gene and being integrated into cotton and tobacco gene group, the salt tolerance of cotton and tobacco plant is improved.The technical scheme is that application of the cotton class calcineurin B subunit gene GhCBL1 in cotton and tobacco breeding.The present invention also provides the plant expression vectors of improvement cotton and tobacco seed, protein including cotton class calcineurin B subunit gene GhCBL1 coding, or the element of the protein is expressed, or express the carrier of the protein, or express the host cell of the protein.The salt tolerance of cotton and tobacco can be improved in cotton class calcineurin B subunit gene GhCBL1 of the invention, provides a kind of new selection for cotton and tobacco breeding.

Description

Cotton class calcineurin B subunit gene GhCBL1 is in cotton and tobacco breeding Using

Technical field

The invention belongs to field of plant genetic project technology, and in particular to cotton class calcineurin B subunit gene Application of the GhCBL1 in cotton and tobacco breeding.

Background technique

China is that one of maximum Cotton Production and country of consumption, Cotton Production are occupied very heavy in national economy in the world The status wanted.But the development with cotton textiles industry with the improvement of people's living standard in our country, the production energy of Cotton in China Power is no longer satisfied domestic demand.China is salt-soda soil big country, occupies the in the country of saline alkali land area row's top 10 Three.Chinese salt-soda soil is distributed in 17 provinces and regions including northwest, northeast, North China and coastal region, saline-alkali wasteland and influence arable land The salt-soda soil gross area be more than 500,000,000 mu, wherein accounting for 10% or more the Chinese arable land gross area with Potential Agricultukal Productivity.Although cotton Flower has the ability of certain saline-alkali tolerant, studies have shown that, salt ionic concentration is higher than under conditions of 0.4% in the soil, cotton Colored sprouting and growth of seedling will receive inhibition (Liu Jian light etc., 2010).If can be further improved the salt tolerance of cotton, so that it may A large amount of salt-soda soil is become available resource, increase the cultivation of cotton under the premise of avoiding seizing arable land with cereal crops Kind area, to improve the yield of cotton.

With increased population and Cultivated Land Area Decrease, in order to guarantee grain security, increasing output of cotton must be avoided " with grain Strive ground ".China possesses a large amount of salt-soda soil, and the salt resistance ability for improving cotton has important meaning to effective use saline alkali land resource Justice.The soil salinization is a global resource and ecological problem.Currently, global saline soil ground area up to 9.5 × 108hm², Chinese about 200,000,000 hm of saline soil ground area².The salination of soil is to limit a key factor of crop yield, It is that world agriculture produces major issue anxious to be resolved (Ma etal., 2011).To solve this problem, on the one hand, need Long-term improvement is carried out to salt-affected soil；On the other hand, need to cultivate the crop new product for adapting to the height salt tolerant of salt-affected soil Kind.

Cotton produces the response mode of a variety of environment stresses during long-term evolution, and calcium signal approach is One of them.Ca²⁺Signal passes through the calcium sensing element containing EF structural domain (elongation factor-hand, EF-hand) (calcium sensor) carries out signal transduction (Day et al., 2002).According to the number of EF-hand structural domain, composition side The homology of formula and amino acid sequence, the calcium ion receptor containing EF-hand structural domain are broadly divided into three classes in plant: calcium from Sub- dependent form protein kinase (Ca²⁺Dependent protein kinase, CDPKs), calmodulin (calmodulin, CaM) With calcineurin B subunit albuminoid (calcineurim B-like proteins, CBL) (Reddy et al., 2011).After cell is by extracellular or intracellular stimulation, Ca in cytoplasm²⁺From cells such as endoplasmic reticulum, mitochondria, chloroplaset and vacuoles Discharge in device, then combined with intracellular receptor calmodulin (calmodulin, CaM) or CaM related protein, further by with Intracellular a variety of enzymes or protein are combined to adjust cell physiological biochemical process (Dodd et al., 2010).

Calcineurin is Ca²⁺The heterodimer phosphoprotein phosphatase of/CaM dependence, and find so far it is unique by Ca²⁺The serine/threonine protein matter phosphatase adjusted with calmodulin (calmodulin, CaM).Calcineurin is by A, B two A subunit composition, the A subunit of calcineurin are catalytic subunit, and B subunit is to adjust subunit, belong to PP2B albuminoid phosphatase. Some researches show that, the extraneous arids of arabidopsis cbl1 afunction mutant strain versus wild type plant pair, salt stress and low Temperature is more sensitive, and plant pair can be improved in overexpression calcineurin B subunit encoding homologous genes AtCBL1 in arabidopsis The tolerance of salt stress illustrates that CBL1 is a important regulating and controlling factor (Wang et of the arabidopsis to extraneous stress response al.,2007).On this basis, we have studied derive from the endogenous calcineurin B subunit gene GhCBL1 of cotton to raising The effect of tobacco and cotton to the tolerance of salt stress obtains the higher crop of salt tolerance improved by technique for gene engineering Material.

Summary of the invention

It is an object of the invention to provide a kind of new selection for cotton and Tobacco Salt breeding.A further object of the present invention It is to provide the preparation method of transgene cotton and tobacco containing above-mentioned plant expression vector.

In order to solve the above technical problems, present invention employs following scheme:

Application of the cotton class calcineurin B subunit gene GhCBL1 in cotton and tobacco breeding.

Further, the application is cotton class calcineurin B subunit gene GhCBL1 resistance in raising cotton plants Application in salt.

Further, the application is cotton class calcineurin B subunit gene GhCBL1 resistance in raising tobacco plant Application in salt.

Further, the cDNA of the cotton class calcineurin B subunit gene GhCBL1 has SEQ ID NO.1 institute The nucleotide sequence shown.

Further, the protein of cotton class calcineurin B subunit gene GhCBL1 coding has SEQ ID Amino acid sequence shown in NO.2.

A kind of plant expression vector for improveing cotton and tobacco seed, including cotton class calcineurin B subunit gene The protein of GhCBL1 coding, or the element of the protein is expressed, or express the carrier of the protein, or express the protein Host cell.

Further, the element of the described expression protein from 5 ' -3 ' directions successively include constitutive promoter p35S, Cotton class calcineurin B subunit gene GhCBL1, terminator.

The invention has the benefit that

Present invention employs the constitutive promoter p35S for deriving from cauliflower mosaic virus (CaMV).The present invention improves cigarette The method of careless plant salt tolerance: the constitutive expression cotton class calcineurin B subunit gene GhCBL1 in tobacco plant, to increase Add the expression quantity of adversity gene in tobacco plant, it is final to improve Tobacco Salt.Meanwhile the present invention passes through constitutive expression cotton Class calcineurin B subunit gene GhCBL1, can be improved the salt tolerance of cotton plants.

Experiment results proved, the plant strain growth of 35S:GhCBL1 transgene tobacco and development are normal, can normally blossom and bear fruit, Growth of seedling and wild type control no significant difference；Corresponding transgenic cotton plant fibrous fracture specific strength is declined slightly, and is planted Plant shape state, hundred grain weight, fiber production and other qualities and wild type control also no significant difference.This shows overexpression GhCBL1 has not significant impact the growth and development of tobacco and cotton plants.35S:GhCBL1 transgene tobacco seedling is being added with Main root elongation, the growth of root hair and true leaf developmental condition are superior to wild type in the NaCl culture medium of 150mM and 200mM.Transgenosis Cotton plants growth conditions in the soil containing 4g/kg and 6g/kg NaCl are better than WT lines.It is seeped in transgenic plant The content of saturating Auto-regulator proline increased, and also have with the expression quantity that plant stress-resistance adjusts related gene different degrees of upper It adjusts.

The result shows that the tolerance of plant pair salt stress can be improved in overexpression GhCBL1 in cotton and tobacco. The present invention provides a kind of new selection for tobacco and salt tolerance of cotton breeding, and the present invention also provides a kind of improvement cotton and tobaccos The plant expression vector of seed.The method of the invention is simple and easy to do, significant effect, can be improved cotton and tobacco seed salt tolerant Property, improve the economic benefit of cotton and tobacco.

Detailed description of the invention

Fig. 1: the sequence analysis of upland cotton class calcineurin B subunit gene (GhCBL1)

Fig. 2: upland cotton GhCBL1 gene is in upland cotton different tissues and the expression quantity of ovule each stage of development

Fig. 3: the building flow chart of upland cotton 35S:GhCBL1 expression vector

Carrier main element mark, gus:nptII: β-glucose neuraminidase merge base with neomycin phosphotransferase Cause；Nos: terminator；Kanamycin: kalamycin resistance gene；P35S: the plant of cauliflower mosaic virus (CaMV) is derived from Object constitutive promoter；CaMV35S poly A:35S terminator；T-Border:T-DNA is inserted into boundary.

Fig. 4: T₀For the expression quantity detection of GhCBL1 gene in transgenic tobacco leaf

Mark, CBL-5, CBL-3, CBL-12, CBL-18, CBL-14, CBL-9, CBL-6, CBL-10, CBL-13 and CBL- 16: different tobacco T₀For transgenic line, WT: wild-type tobacco.

Fig. 5: Tobacco Salt analysis

Fig. 6: T₀For the expression quantity detection of GhCBL1 gene in transgene cotton blade

Fig. 7: salt tolerance of cotton analysis

Specific embodiment

Below with reference to examples and drawings, the present invention is described in further detail, but embodiments of the present invention are not It is limited to this.

Reagent chemicals in present example do not do illustrate be it is common commercially available, MATERIALS METHODS does not illustrate Refer to " Molecular Cloning:A Laboratory guide " (Sambrook and Russell, 2001).Primer sequence used in embodiment is shown in Table 1.

1 primer sequence of table

Primer	Sequence 5 ' -3 '	Sequence number
			GhCBL1cDNA sequence amplification primer 1	GGATCCATGGGCTGCTTTCAATCTAAAG	SEQ ID No.4
GhCBL1cDNA sequence amplification primer 2	GTCGACTCATGTGGCAAACTCATCAACC	SEQ ID No.5
			GhCBL1 quantification PCR primer 1	ATGACATGGATGGAACGGGT	SEQ ID No.6
GhCBL1 quantification PCR primer 2	CGTCCTGATTAACGTCCGCA	SEQ ID No.7
			Histone3-1	GAAGCCTCATCGATACCGTC	SEQ ID No.8
Histone3-2	CTACCACTACCATCATGGC	SEQ ID No.9
			Gh_SOS3-F	GGCTTTATCGAGCGTGAGGA	SEQ ID No.10
Gh_SOS3-R	ATCTCCATTGACGGAGACGC	SEQ ID No.11
			KIN1-F	GCAGCTGGTGCTGGAGCTGGA	SEQ ID No.12
KIN1-R	CTTGTTCAGGCCGGTCTTGT	SEQ ID No.13
			DREB2A-F	CAGCAGGATTCGCTATCTGT	SEQ ID No.14
DREB2A-R	CATCCTTTCCCTCGAGCTGA	SEQ ID No.15
			RD29A-F	GACTGATGAGGTGAAGCCAGA	SEQ ID No.16
RD29A-R	CCAAGTGATTGTGGAGACTCT	SEQ ID No.17
			Gh_Myb108-F	ACGTTTGGATCCCTCGTCTG	SEQ ID No.18
Gh_Myb108-R	TTCAACCCCACCCCATGTTC	SEQ ID No.19

Embodiment 1: the clone of upland cotton class calcineurin B subunit gene (GhCBL1)

The RNA of cotton ovule is extracted using EASYspin plant RNA rapidly extracting kit (Aidlab).Referring to Takara Specification carries out the synthesis of cDNA, the template as GhCBL1 gene cloning.Finally specifically drawing with the gene of design synthesis Object expands the global cDNA sequence of the gene using above-mentioned cDNA as template.Amplification condition is as follows: 10 × PCR buffer 5 μ L, 25mmol/L MgSO of for KOD Plus₄5 μ L, 2mmol/L dNTPs, 2 μ L, primer 1 (5 μm of ol/L) 2 μ L, primer 2 (5 μm of ol/L) 2 μ L, KOD Plus polymerase 1U/ μ L, upland cotton cDNA about 60ng, distilled water complement to 50 μ L.Amplification program Are as follows: 94 DEG C, 2min；94 DEG C, 15sec；56 DEG C, 30sec；68 DEG C, 1.5min；35 circulations.After the completion of amplification, agarose electrophoresis And corresponding DNA band is recycled, the survey of Hua Da company is sent to after being cloned into blunt vector pEASY-Blunt (TransGen Biotech) Sequence verifying.

In ncbi database, the class calcineurin B subunit gene (GhCBL1) from different plant species is selected to carry out same Source property compares.The sequence of participation analysis: BbCNB (GenBank accession number: XP_008600476.1) (beauveria bassiana, Beauveria bassiana), GhCBL1 (GenBank accession number: NP_001313993.1) (cotton, Gossypium Hirsutum), NtCBL1 (GenBank accession number: XP_016451516.1) (tobacco, Nicotiana tabacum), AtCBL1 (GenBank accession number: NP_567533.1) (arabidopsis, Arabidopsis thaliana), (GenBank is logged in AtCBL9 Number: NP_199521.1) (arabidopsis, Arabidopsis thaliana).As shown in Figure 1, GhCBL1 albumen is with these from not Infraspecific albumen is the same, and there is conserved domain Pfam Domain, transmembran helix region, shows institute The cotton GhCBL1 gene of clone encodes class calcineurin B subunit gene.

The recycling of embodiment 2:DNA segment, vector construction, Escherichia coli conversion

Under ultraviolet lamp, the Ago-Gel block containing target fragment is cut with clean blade, according to kit (Aidlab) Method recycle corresponding DNA fragmentation.Vector construction process is shown in Fig. 3.All restriction enzymes are purchased from Roche company, according to Operation instructions operation.

The segment of recycling establishes following linked system with carrier: 10 × T4DNA connect 1 μ L of buffer, 1 μ of vector DNA fragment L, 1 μ L, T4DNA ligase of external source connection product DNA fragmentation, 1 μ L, the linked system of volume to 10 μ L is supplied with distilled water.

Vector DNA fragment and external source connection product DNA fragmentation molar ratio are 1:3,16 DEG C of connection 12h.Connection is produced later Object converts bacillus coli DH 5 alpha.

Entire T-DNA section about 17.8kb of the present invention for the expression vector plasmid of Cotton Transformation, structure are shown in Fig. 3, wrap Include two fusion (gus:nptII), target fragment (GhCBL1) Expression elements of riddled basins and reporter gene.Really Determining riddled basins characteristic can be detected using PCR, and reporter gene gus can be determined by histochemical stain.Screening Whether target fragment, which has been integrated into cotton DNA, to be detected by PCR.Target gene GhCBL1 is built into plant expression and carries The process of body pLGN is shown in Fig. 3.On the basis of p5 skeleton carrier, T-DNA section (region between RB and LB) is substituted for label The track fusion box of gene nptII, and it is respectively added to a LoxpFRT recombination enzyme recognition site at P5 expression cassette both ends, And another by CaMV35S-P control expression cassette.According to the building flow chart of expression vector, using corresponding restricted interior Enzyme cutting digestion constructs the specific expression carrier of GhCBL1 gene according to above-mentioned Ligature.

Embodiment 3: the genetic transformation of Agrobacterium and tobacco and cotton

1. the plant expression carrier plasmid of building is imported Agrobacterium LBA4404 with electrization.

With reference to Bio-RAD MicroPulser instruction manual book, above-mentioned plant expression vector is led by Electroporation conversion Enter Agrobacterium LBA4404.

2. excess forward direction expression vector is integrated into tobacco gene group

Using the leaf disc transformation method (Horsch, et al., 1985) of mediated by agriculture bacillus, genetic transformation is carried out to tobacco, it will The transgenic tobacco plant of acquisition is screened by the method that GUS is dyed, and GUS stained positive plant is planted in greenhouse In, Routine Management collects seed sowing and carries out Salt Tolerance Analysis after mature.

3. specific expression carrier is integrated into upland cotton genome

Genetic transformation (Luo et al., 2007) is carried out to cotton using the method for mediated by agriculture bacillus, acquisition is turned into base Because cotton plants are screened by the method that GUS is dyed, the seedling of GUS stained positive is placed in clear water, 22 DEG C of cultures 1 Transplanting carries out normal management into greenhouse after week.

Embodiment 4: the extraction of tobacco and each tissue RNA of upland cotton and quantitative PCR analysis

The RNA of tobacco and each tissue of cotton is extracted using EASYspin plant RNA rapidly extracting kit (Aidlab). The reverse transcription of mono- chain of cDNA is carried out referring to RevertAid First Strand cDNA Synthesis Kit (MBI) specification, As quantitative RT PCR analysis template.Using iQ SYBR Green Supermix (BIO-RAD) reagent analysis target gene Relative expression quantity.Internal standard gene selects the GhHIS1 gene (AF024716) of upland cotton and the Ncactin gene of tobacco, and primer is Histone3-1(5’-GAA GCC TCATCG ATA CCG TC-3’)、Histone3-2(5’-CTA CCA CTA CCA TCA TGG C-3 ') and ACT1 (5 ' GAT GGT GTC AGC CAC ACT GTC 3 '), ACT2 (5 ' ATG CTGCTA GGA GCC AGT GC 3').GhCBL1 gene expression amount detects the primer are as follows: GhCBL1 quantification PCR primer 1 (5 '- ATGACATGGATGGAACGGGT-3 ') and GhCBL1 quantification PCR primer 2 (5 '-CGTCCTGATTAACGTCCGCA-3 ').Remaining The quantification PCR primer of gene is shown in sequence table.Amplification condition are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 20s, 56 DEG C of annealing 20s, 72 DEG C of extension 30s, 40 circulations.Target gene and interior target ratio in each material are calculated, has both obtained target gene in Different Organs Relative expression quantity in tissue, is shown in Fig. 2 and Fig. 4.

Using real-time quantitative RT-PCR detection GhCBL1 gene in the stem of Ji cotton 14 (WT), leaf, the different tissues and not such as spend Expression in ovule and fiber of the same period, as shown in Fig. 2, GhCBL1 gene is sent out in each nutrition organs and ovule and fiber The different times educated have expression, without apparent spatial and temporal expression specificity.

Construct the expression vector pLGN-35S:GhCBL1 of CaMV35S promoter regulation GhCBL1.Utilize mediated by agriculture bacillus Genetic transformation will be in 35S:GhCBL1 overexpression vector introduction tobacco.It is identified by antibiotic-screening and GUS tissue staining Method filters out transgenic tobacco plant.The transgene tobacco that screening is obtained plants the inverted third in greenhouse nutritive cube The expression of blade extraction RNA analysis GhCBL1.See Fig. 4, qRT-PCR is the results show that GhCBL1 can be just in tobacco Often expression.In GhCBL1 transgenic line, the table of 10 strains of 35S:GhCBL1 transgene tobacco and wild type progress GhCBL1 Up to analysis, as a result CBL-5 expression is higher, improves about 900 times than wild type.

35S:GhCBL1 is transferred in Ji cotton 14 by Agrobacterium tumefaciens-mediated Transformation method, by turning for GUS stained positive Gene cotton plants are planted in greenhouse, extract the RNA of transgene cotton seedling plants leaf third from the bottom for measuring corresponding base The expression of cause.As shown in fig. 6, qRT-PCR is the results show that GhCBL1 gene in GhCBL1-12 and GhCBL1-36 plant Expression is higher.

Embodiment 5:GhCBL1 transgene tobacco Salt Tolerance Analysis

As shown in Figure 5A, compared with wild-type tobacco, there is no obviously becoming for the phenotype of transgene tobacco GhCBL1 plant Change.Transgene tobacco can normally blossom and bear fruit, and plant forms and wild type do not have notable difference.

As shown in Figure 5 B, culture transgene tobacco is sprouted on the MS culture medium without NaCl, main root is long after counting 15d, The long main root of the main root of wild-type tobacco a length of 5.39 ± 1.25cm, CBL-5 and CBL-3 is respectively 5.79 ± 0.96cm and 5.90 ± 1.27cm, no significant change compared with wild type show that transgene tobacco can normal germination and growth.Containing 100mM On the MS culture medium of NaCl grow 15d after, the main root of wild-type tobacco a length of 2.26 ± 1.43cm, transgene tobacco CBL-5 and The long main root of CBL-3 transformant is respectively 3.33 ± 0.64cm and 5.03 ± 0.87cm, obvious compared with the main root of wild-type tobacco It is elongated.After growing 15d on the MS culture medium containing 200mMNaCl, the root long of CBL-5 and CBL-3 transformant compares wild-type tobacco Longer, and the survival rate of each transgenic tobacco plant is above WT lines, wild-type tobacco has been wilted flavescence, transgenosis Tobacco seedling is also survived, and only the elongation of main root is suppressed.The result shows that in tobacco overexpression cotton source calcium tune The salt tolerance of tobacco plant can be improved in calcineurin B subunit homologous gene GhCBL1.

The measurement of embodiment 6:GhCBL1 transgene cotton agronomic shape

Salt tolerance of cotton analysis:

Sufficient amount of full cotton seeds are chosen with 1 ‰ H₂O₂Aqueous solution soaking 12h, be uniformly layered on and be placed with multilayer It in the culture dish of wet filter paper, then covers multilayer and moistens filter paper, culture dish is placed in 30 DEG C of constant temperature dark culture casees, is sprouted to seed Hair, when hypocotyl elongation is to 2~3cm, move to water planting in the plastic cup equipped with water (28 ± 2 DEG C, relative humidity 85%, when illumination Between 13h/d), periodically keep the skin wet.Cotton Seedling-Growth chooses seedling similar in growth conditions, is transferred to and is equipped with to 2 leaf phases In the plastic culture cup of the aqueous solution of 0mM and 200mM NaCl (28 ± 2 DEG C, relative humidity 85%, light application time 13h/d).With The aqueous solution processing of 0mM NaCl is control, using 200mM NaCl aqueous solution as salt stress processing, replacement etc. every other day between experimental period The solution of amount chooses corresponding material for taking pictures and analyzing after 10d.As shown in Figure 7 A, non-transgenic type Young Cotton The cotyledon and true leaf of seedling have all been wilted, and the cotyledon and true leaf of transgene cotton do not occur wilting or less wilting, show excess The salt resistance ability of cotton seedling can be improved in expression GhCBL1.

Three control (0g/kg), moderate (4g/kg), intensity (6g/kg) gradients are provided with using fluid-tight flowerpot come mould Cotton seedling after quasi- salt-soda soil plantation transplanting, observation plant field growing way discovery WT and transgenosis indifference under control case It is different, but under salt stress, the plant height of transgene cotton, leaf blade size, blade quantity is all larger than WT, as shown in Fig. 7 B.

The variation in transgenic cotton flower maturity plant leaf with stress response related gene expression level is had detected, with clear Influence of the overexpression GhCBL1 to cotton stress response approach in cotton.In gene detected, DREB2A can be rung Answer ABA and osmotic stress (Liu et al., 1998)；RD29A and KIN1 can be lured by cold, arid, salt stress and ABA Lead (Kurkela and Borg-FrancK, 1992；Tahtiharju et al.,1997)；MYB108 is calcium ion/calmodulin The myb transcription factor of dependence can respond the environment stresses such as arid, salt stress (Yoo et al., 2005)；SOS3 is in plant Salt ion response element (Chinnusamy et al., 2004；Mahajan et al.,2008).As seen in figure 7 c, qRT-PCR The results show that the expression of gene relevant to stress response has compared with wild type in the transgene cotton of normal growth It is improved.In the case where the induction of no external environment, in cotton when overexpression GhCBL1, these and Stress response The expression of relevant gene raises, and shows that transgenic cotton plant has adaptation external environment on gene expression dose The ability of stress.

Plant can activate the expression of related gene after by environment stress, make the relevant substance hair of stress in plant Changing is to cope with extraneous variation.Proline is one of important component of phytoprotein, and can be wide with free state It is general to be present in plant.Since proline hydrophily is extremely strong, in the metabolic process in histocyte, have reduce freezing point and Prevent the effect of cell dehydration.Therefore (arid, saline and alkaline, hot, cold, jelly), the intracorporal proline content of plant under adverse environmental factor It can dramatically increase, the content of proline reflects the resistance of plant to a certain extent in plant, therefore measures proline Content is to evaluate a physical signs of plant drought resistance.

Cotton leaf similar in greenhouse normal growth and growth conditions is chosen, osmotic field dried meat ammonia in fresh blade is extracted Acid, as illustrated in fig. 7d, in the case where no external environment stress, proline content is compared with control group cotton in each transgenic line Increase, wherein in CBL-1, CBL-12 and CBL-36 blade proline content be 13.68 ± 0.01 μ g/g, 13.49 ± 67.4% and 65.1% He has been respectively increased in 0.01 μ g/g and 13.95 ± 0.01 μ g/g, 8.17 ± 0.01 μ g/g compared with control group 68.2%.The result shows that overexpression GhCBL1 gene in cotton, the content of osmotic field proline is increased, so that its is resistance to Salt is improved.

The measurement that levant cotton seed refers to, ginning outturn, clothing refer to:

By the unginned cotton harvested machine lint, the son for counting transgene cotton and non-transgenic cotton refers to, ginning outturn, clothing Refer to.100 unginned cottons are selected, after cotton ginning, weigh 100 cotton fiber weight (also referred to as clothing refers to) and 100 seed weights (also referred to as respectively Son refers to), measure the ginning outturn (weight of cotton fiber/cotton fiber+cottonseed gross weight) of cotton.Each transgenic plant chooses 300 Unginned cotton, 3 repetitions, statistical data are shown in Table 2.

The son of 2 35S:GhCBL1 transgene cotton of table and wild type cotton refers to, ginning outturn and clothing refer to comparison

Gene serial number	Son refers to (g)	Ginning outturn (%)	Clothing refers to (g)
				WT	10.91±0.02	35.42±0.14	5.98±0.01
CBL-1	9.57±0.24	37.42±0.24	5.72±0.14
				CBL-12	10.10±0.19	36.89±0.15	5.90±0.11
CBL-3	11.18±0.19	36.75±0.18	6.50±0.11
				CBL-2	10.22±0.32	35.92±0.32	5.73±0.18
CBL-36	11.00±0.08	37.28±0.24	6.54±0.05

As shown in table 2, control group refers to that for 10.91 ± 0.02g, transgenosis is in 9.57 ± 0.24g (CBL-1) to 11.18 Between ± 0.19g (CBL-3), variation is little compared with the control.The result shows that overexpression GhCBL1 gene pairs cotton in cotton Flower seed size and weight do not influence significantly.Fiber yield is the percentage that fiber accounts for unginned cotton weight, it may be assumed that ginning outturn (%)= Fibre weight/(fibre weight+seed weight) × 100.Statistical result showed, the ginning outturn of non-transgenic control is 35.42 ± 0.14%, transgene cotton is between 35.92 ± 0.32% (CBL-2) to 37.42 ± 0.24% (CBL-1), with control group phase Than slightly raising, but change unobvious.It is weight that every hundred cottonseeds generate fiber that clothing, which refers to, the results show that the clothing of control group refers to For 5.98 ± 0.01g, the clothing of transgene cotton refers between 5.72 ± 0.14g (CBL-1) to 6.90 ± 0.11g (CBL-2), with Control group compares no notable difference.The result shows that overexpression GhCBL1 gene pairs cotton seeds generate fiber in cotton Ability do not influence significantly.

Cotton fiber quality detection:

As shown in table 3, fiber quality testing result is shown, the fibre length of transgene cotton is arrived in 27.53 ± 0.19mm Between 29.3 ± 0.50mm, do not changed significantly compared with 29.53 ± 0.42mm of control group.Mic value is as measurement cotton The overall target of flower fibre maturity and fineness, in a certain range, mic value is lower, and expression fiber is thinner.Testing result is aobvious Show, the mic value of transgene cotton is compared with the control group without significant change.But the fibre strength of transgene cotton is more right It is declined slightly according to group.From the point of view of fibre uniformity and elongation, transgene cotton with compare between there is no notable difference.Overall knot Fruit shows other than fibrous fracture specific strength is declined slightly, in cotton overexpression GhCBL1 gene pairs cotton fiber quality Other indexs there is no significant impact.

The comparison of the fiber quality of 3 35S:GhCBL1 transgene cotton of table and wild type cotton

Above-described embodiment shows the method that the present invention improves cotton and tobacco seed character, can be realized endogenous regulation kind Son development, overexpression GhCBL1 has not significant impact the growth and development of plant in cotton and tobacco, and cotton can be improved With tobacco plant to the tolerance of salt stress.

The above described is only a preferred embodiment of the present invention, be not intended to limit the present invention in any form, according to According to technical spirit of the invention, within the spirit and principles in the present invention, it is to the above embodiments it is any it is simple modification, Equivalent replacement and improve etc., fall within the scope of protection of the technical scheme of the present invention within.

SEQUENCE LISTING

<110>Southwest University

<120>application of the cotton class calcineurin B subunit gene GhCBL1 in cotton and tobacco breeding

<130> 2018

<160> 18

<170> PatentIn version 3.3

<210> 1

<211> 642

<212> DNA

<213>artificial sequence

<400> 1

atgggctgct ttcaatctaa agtaacaaga caataccctg gacacgaaga tcctattatt 60

ctagcttctc aaactgcttt tagtgttagt gaagttgaag cactctttga gctattcaag 120

agcataagca gttctgtgat tgatgatggg ctgatcaaca aggaagagtt tcaattagca 180

ctgtttcaga ataggaagaa ggagaacatt ttcgcaaatc ggatttttga tttatttgac 240

gcaaagaaga agggagtcat tgattttggt gactttgttc gagcactcaa tgtcttccat 300

ccaaatgtct cacaagagga caagatgaat tttgcgttta gactctatga catggatgga 360

acgggtttta ttgagcgtaa cgaggtcaag caaatggtga ttgcactgtt atgtgaatct 420

gaaatgaagt tagctgatga aaccattgag gcaattcttg ataagacatt cttggatgcg 480

gacattaatc aggacgggaa gatcgatata tctgaatgga aaaacttcgt ttcccgtaac 540

ccatcactgt tgaaaatcat gacccttcca tacctcaggg atataacaac aacatttcct 600

agctttgttt tccattctga ggttgatgag tttgccacat ga 642

<210> 2

<211> 213

<212> PRT

<213>artificial sequence

<400> 2

Met Gly Cys Phe Gln Ser Lys Val Thr Arg Gln Tyr Pro Gly His Glu

1 5 10 15

Asp Pro Ile Ile Leu Ala Ser Gln Thr Ala Phe Ser Val Ser Glu Val

20 25 30

Glu Ala Leu Phe Glu Leu Phe Lys Ser Ile Ser Ser Ser Val Ile Asp

35 40 45

Asp Gly Leu Ile Asn Lys Glu Glu Phe Gln Leu Ala Leu Phe Gln Asn

50 55 60

Arg Lys Lys Glu Asn Ile Phe Ala Asn Arg Ile Phe Asp Leu Phe Asp

65 70 75 80

Ala Lys Lys Lys Gly Val Ile Asp Phe Gly Asp Phe Val Arg Ala Leu

85 90 95

Asn Val Phe His Pro Asn Val Ser Gln Glu Asp Lys Met Asn Phe Ala

100 105 110

Phe Arg Leu Tyr Asp Met Asp Gly Thr Gly Phe Ile Glu Arg Asn Glu

115 120 125

Val Lys Gln Met Val Ile Ala Leu Leu Cys Glu Ser Glu Met Lys Leu

130 135 140

Ala Asp Glu Thr Ile Glu Ala Ile Leu Asp Lys Thr Phe Leu Asp Ala

145 150 155 160

Asp Ile Asn Gln Asp Gly Lys Ile Asp Ile Ser Glu Trp Lys Asn Phe

165 170 175

Val Ser Arg Asn Pro Ser Leu Leu Lys Ile Met Thr Leu Pro Tyr Leu

180 185 190

Arg Asp Ile Thr Thr Thr Phe Pro Ser Phe Val Phe His Ser Glu Val

195 200 205

Asp Glu Phe Ala Thr

210

<210> 3

<211> 28

<212> DNA

<213>artificial sequence

<400> 3

ggatccatgg gctgctttca atctaaag 28

<210> 4

<211> 28

<212> DNA

<213>artificial sequence

<400> 4

gtcgactcat gtggcaaact catcaacc 28

<210> 5

<211> 20

<212> DNA

<213>artificial sequence

<400> 5

atgacatgga tggaacgggt 20

<210> 6

<211> 20

<212> DNA

<213>artificial sequence

<400> 6

cgtcctgatt aacgtccgca 20

<210> 7

<211> 20

<212> DNA

<213> Unknown

<220>

<223>artificial sequence

<400> 7

gaagcctcat cgataccgtc 20

<210> 8

<211> 19

<212> DNA

<213>artificial sequence

<400> 8

ctaccactac catcatggc 19

<210> 9

<211> 20

<212> DNA

<213>artificial sequence

<400> 9

ggctttatcg agcgtgagga 20

<210> 10

<211> 20

<212> DNA

<213>artificial sequence

<400> 10

atctccattg acggagacgc 20

<210> 11

<211> 21

<212> DNA

<213>artificial sequence

<400> 11

gcagctggtg ctggagctgg a 21

<210> 12

<211> 20

<212> DNA

<213>artificial sequence

<400> 12

cttgttcagg ccggtcttgt 20

<210> 13

<211> 20

<212> DNA

<213>artificial sequence

<400> 13

cagcaggatt cgctatctgt 20

<210> 14

<211> 20

<212> DNA

<213>artificial sequence

<400> 14

catcctttcc ctcgagctga 20

<210> 15

<211> 21

<212> DNA

<213>artificial sequence

<400> 15

gactgatgag gtgaagccag a 21

<210> 16

<211> 21

<212> DNA

<213>artificial sequence

<400> 16

ccaagtgatt gtggagactc t 21

<210> 17

<211> 20

<212> DNA

<213>artificial sequence

<400> 17

acgtttggat ccctcgtctg 20

<210> 18

<211> 20

<212> DNA

<213>artificial sequence

<400> 18

ttcaacccca ccccatgttc 20

Claims

1. application of the cotton class calcineurin B subunit gene GhCBL1 in cotton and tobacco breeding.

2. cotton class calcineurin B subunit gene GhCBL1 according to claim 1 is in cotton and tobacco breeding Using, it is characterised in that: the application is that cotton class calcineurin B subunit gene GhCBL1 is improving cotton plants salt tolerant Application in property.

3. cotton class calcineurin B subunit gene GhCBL1 according to claim 1 is in cotton and tobacco breeding Using, it is characterised in that: the application is that cotton class calcineurin B subunit gene GhCBL1 is improving tobacco plant salt tolerant Application in property.

4. described in any item cotton class calcineurin B subunit gene GhCBL1 are in cotton and tobacco according to claim 1~3 Application in breeding, it is characterised in that: the cDNA of the cotton class calcineurin B subunit gene GhCBL1 has SEQ ID Nucleotide sequence shown in NO.1.

5. described in any item cotton class calcineurin B subunit gene GhCBL1 are in cotton and tobacco according to claim 1~3 Application in breeding, it is characterised in that: the protein of the cotton class calcineurin B subunit gene GhCBL1 coding has Amino acid sequence shown in SEQ ID NO.2.

6. a kind of plant expression vector for improveing cotton and tobacco seed, which is characterized in that sub- including cotton class calcineurin B The protein of base gene GhCBL1 coding, or the element of the protein is expressed, or express the carrier of the protein, or express the egg The host cell of white matter.

7. the plant expression vector of improvement cotton and tobacco seed according to claim 6, it is characterised in that: the cotton The protein of flower class calcineurin B subunit gene GhCBL1 coding has amino acid sequence shown in SEQ ID NO.2.

8. the plant expression vector of improvement cotton and tobacco seed according to claim 6, it is characterised in that: the cotton The cDNA of flower class calcineurin B subunit gene GhCBL1 has nucleotide sequence shown in SEQ ID NO.1.

9. the plant expression vector of improvement cotton and tobacco seed according to claim 6, it is characterised in that: the table Up to the element of the protein from 5 ' -3 ' directions successively include constitutive promoter p35S, cotton class calcineurin B subunit gene GhCBL1, terminator.