CN108753794A - Plant stem holds juice controlling gene Dry and its coding albumen - Google Patents

Abstract

The present invention describe one in plant regulate and control cane hold the Dry genes nucleotide sequence of juice and its protein amino acid sequence of coding, belong to gene engineering technology field.The nucleotide sequence and protein have the function of that plant holds juice, juice, which is held, for genetic engineering Crop Improvement stalk provides important candidate gene, it can be used for manufacturing for bio-ethanol, syrup and ensilage, there is huge application and economic value.

Description

Plant stem holds juice controlling gene Dry and its coding albumen

Technical field

The present invention relates to a kind of plant stems to hold juice controlling gene Dry and its coded protein sequence, belongs to gene work Journey field.

Background technology

Water regime in plant is related to many important Physiological Activities of Plants, but at present for moisture in plant The research of interior absorption, storage and transhipment is not still deep enough.Sugar grass can be used for biology as a kind of new energy crop Ethyl alcohol, syrup and ensilage are manufactured.In its many influence factor, moisture is a particularly important side Face.The completion successively of sorghum gene order-checking and again examining order learns to do piecewise analysis by forward genetics for us and research is planted The mechanism that object regulates and controls its internal water provides important basis.

Invention content

The present invention provide it is a kind of regulation and control Sorghum Stalk hold juice gene Dry DNA sequence dna, cDNA sequence and the gene The normal expression of the amino acid sequence of coded functional protein, the gene can be such that moisture in plant significantly reduces.

The present invention first goal of the invention be：A kind of regulation and control plant is provided and holds the new gene Dry of juice, feature exists In, be selected from following DNA molecular 1) or 2) or 3) or 4) or 5)：

1) DNA molecular shown in SEQ ID NO.1 (cloning the genomic DNA from place of china sorghum variety Ji2731)： The DNA molecular is made of 4155 nucleotide, is first exon, 170-1571 from the nucleotide of 5 ' end 1-169 Position nucleotide is First Intron, and 1572-1837 nucleotide are second exon, 1838-3678 nucleotide For second introne, 3679-4155 nucleotide are third exon；

2) DNA molecular shown in SEQ ID NO.2 (cloning the cDNA from place of china sorghum variety Ji2731)；

3) on the bases SEQ ID NO.1 by one to several bases replace and or one to several bases insertion With or the nucleotide sequence of missing and large fragment be inserted into missing displacement inversion formed and can influence plant fecundity DNA molecular；

4) 0.1 × SSPE (or 0.1 × SSC), 0.1% (w/v) SDS solution in, hybridize and wash under the conditions of 65 DEG C Film, can hybridize and the DNA molecular of coded plant moisture regulation GAP-associated protein GAP with the DNA molecular of SEQ ID NO.2；

5) to the DNA molecular of SEQ ID NO.2 with 70% or more homology and the related egg of coded plant moisture regulation White DNA molecular.

The present invention second goal of the invention be：The phase that the encoded regulation and control plant stem of said gene holds juice is provided Close protein.

In one embodiment, the gene code it is following 1) or 2) described in protein：

1) protein that amino acid sequence shown in the SEQ ID NO.3 of sequence table forms；

2) by the SEQ ID NO.3 of sequence table by one or several amino acid residues substitution, and/or missing and/ Or the protein of addition and the related activity with influence plant moisture content.

The present invention third goal of the invention be：Recombinant expression carrier containing the gene is provided, expression cassette, turns base Because of cell line or recombinant bacterium.

4th goal of the invention of the invention be：Said gene is provided in the purposes for transgene improvement crop.

In one embodiment, the gene holds juice for regulating and controlling plant haulm, and stalk moisture is made significantly to drop It is low.

In a specific embodiment, the gene makes the reduction of plant content of cellulose, hemicellulose level liter It is high.

In a more specifical embodiment, the crop include but not limited to sorghum, corn, rice, millet and False bromegrass.

5th goal of the invention of the invention be：Provide a kind of ortholog base obtaining Dry genes in other plants The method of cause, and corn (Zea mays), broomcorn millet (Panicum hallii), switchgrass using this method acquisition (Panicum virgatum), green bristlegrass (Setaria viridis), millet (Setaria italica), false bromegrass The amino acid sequence of (Brachypodium distachyon) and rice (Oryza sativa).

Compared with prior art, the present invention has following advantageous effect：Plant stem provided by the invention holds juice tune Control gene Dry directly participates in the transport process of plant internal water, after the expression of the gene is suppressed, plant internal water Content increases, and vascular bundle cell development is abnormal.By Plant Biotechnology approach, the present invention is in bio-ethanol, syrup and blueness Significant role will be all played in storage the manufacturing of feed.

Term defines

Term " plant stem holds juice controlling gene " refers to one section of nucleotide sequence with coding albumen ability, the sequence The special coding of row has the protein active polypeptide of regulation and control plant moisture content, such as the 1-912 nucleotide of SEQ ID NO.2 Sequence and its degenerate sequence.

Term " degenerate sequence " refers in 1-912 nucleotide sequences of encoder block of SEQ ID NO.2, there is one The sequence that the degenerate codon that a or multiple codons are encoded same amino acid generates after replacing.Due to codon Degeneracy, so also can down to about 70% degenerate sequence with the 1-912 nucleotide sequence homologies of SEQ ID NO.2 Encode out the encoded amino acid sequences of SEQ ID NO.2.

" plant stem holds juice controlling gene ", further include under moderate stringency, it is more preferably stringent in height Under the conditions of can be with the nucleotide sequence of the nucleotide sequence hybridization of SEQ ID NO.2.Wherein, medium stringency condition can be 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS (w/v) solution in, hybridize and wash the condition of film under the conditions of 65 DEG C.

" plant stem holds juice controlling gene ", further include in SEQ ID NO.2 from 1-912 nucleotide The homology of sequence at least 70%, preferably have at least 80%, 82%, 85%, 86%, 88%, 89% homology, more preferably Ground have at least 90%, 91%, 92%, 93%, 94% homology, most preferably have at least 95%, 96%, 97%, 98%, The nucleotide sequence of 99% homology.

" plant stem holds juice controlling gene " further includes that can encode to have to hold juice gene with natural cane Dry identical function albumen, SEQ ID NO.2 open reading frame sequences variant form.These variant forms include (but simultaneously It is not limited to)：Missing, insertion and/or the substitution of 1 or several nucleotide, and several in 5 ' or 3 ' end additions (is usually 60 Within a, within preferably 30, more preferably it is within 10, is most preferably within 5) nucleotide.

" plant stem holds juice controlling gene " further includes that can translate a kind of ammonia for having regulation and control moisture Base acid sequence, such as the amino acid sequence of SEQ ID NO.3.Such amino acid sequence further includes having to hold juice with natural cane The variant form of the SEQ ID NO.3 of albumen identical function.These variant forms include (but being not limited to)：1 or several ammonia Missing, insertion and/or the substitution of base acid, and C-terminal/or N-terminal addition it is one or several (be usually 20 within, compared with It is more preferably within 5 within 10 to be goodly) amino acid.In the art, with amino acid similar in performance into When row substitution, the function of protein is not usually changed；It is usual that one or several amino acid are added in C-terminal and/or N-terminal The function of protein will not be changed.

In addition, the nucleotide full length sequence or its segment of " plant stem holds juice controlling gene " usually can be with It is obtained with PCR amplification method, recombination method or artificial synthesized method.For PCR amplification method, can have according to disclosed in the present embodiment It closes nucleotide sequence, especially open reading frame sequence and carrys out design primer, the commercially available libraries cDNA are used in combination or by people in the art As template, amplification obtains related sequence in the libraries cDNA prepared by conventional method known to member.Once obtaining related sequence Row, can obtain related sequence in large quantity with recombination method.It is typically cloned into carrier, then cell transformation etc. is normal Rule method isolated related sequence from the host cell of proliferation.Implement in addition, can also be introduced mutation by chemical synthesis In example protein sequence.Other than being generated with recombination method, solid phase technique also can be used in the segment of embodiment albumen, by directly closing It is produced at polypeptide.Synthetic protein can carry out manually or automatically in vitro, can distinguish chemical synthesis embodiment Then each segment of albumen is chemically connected to generate the protein molecule of overall length.

Description of the drawings

Fig. 1 for no juice and hold juice sorghum culm morphology observation and physiological and biochemical analysis.(a) table in stalk cross section Type observe (white scale represents 1 centimetre), wherein stalk without juice sorghum Ji2731 (left side), stalk hold juice sorghum E-tian (in) And filial generation F1 plant (right side)；(b) stalk without juice and holds juice sorghum from the change of jointing stage to milk stage plant stalk water content Change, NL：Jointing stage (the 9th leaf occurs), FLS：Boot leaf phase, HS：Heading stage, FS：Florescence, AF：Post flowering one week, MS： Milk stage；(c) maturity period Sorghum Stalk water content counts, including stalk holds juice sorghum E-tian without juice sorghum Ji2731, stalk And recombinant inbred lines plant material (stalk exchanges single plant without juice and stalk holds juice and exchanges single plant), bracket inner digital represents single plant Number.(d to n) Sorghum Stalk microexamination.Sorghum Stalk marrow vascular bundle and parenchyma cell semithin section observation, stalk without Juice material Ji2731 (d and e) and cane hold juice sugar grass E-tian (f and g).Sorghum Stalk marrow vascular bundle ultra-thin section is aobvious It is microcosmic to examine, Ji2731 (h) and E-tian (k).The microexamination of vascular bundle surrounding catheter parenchyma cell (with white edge include it is crosscutting Face corresponds to), Ji2731 (i) and E-tian (l).Cell wall thickness microexamination (corresponding with the cross section that white edge includes), Ji2731 (j) and E-tian (m).(n) cell wall thickness of surrounding catheter parenchyma cell measures.(o and p), stalk is without juice and holds The stalk proximate analysis of juice sorghum.(o) content of cellulose measures；(p) xylan (hemicellulose main constituents) content It measures.AIR, alcohol-insoluble substances.Bilateral t, which is examined, determines statistical significance, * P < 0.05, * * P < 0.01.

Fig. 2 is Dry gene map based clonings.(a) Dry genes just position, and Ji2731 (no juice) and E-tian (having juice) is carried out The sites Dry are navigated to height by reciprocal cross, the exchange single plant and 30 pairs of PAV and SSR molecular labelings of 198 F2 segregating populations of selection In the section of No. 6 chromosome 3.25-Mb of fine strain of millet；(b) Dry genes finely positioning utilizes the recombinant inbred lines of 3343 F2~6 By the physical distance section (between M10-M11 labels) of Dry genes finely positioning to 42-kb, the long rectangle of white represents E-tian Genotype, the long rectangle of black represent Ji2731 genotype, and white blackens a little long rectangle and represents heterozygous genotypes；(c) Dry genes Functional gene schematic diagram in the physical map section of place, includes four genes in section, and pink colour frame represents candidate gene Sobic.006g147400。

Fig. 3 is the Genomic change analysis nearby of Dry genes and Dry gene expression analysis.(a) 16 couples of PCR are designed to draw Object expands Dry upstream region of gene 7-kb to the genome sequence in downstream regions 2-kb, finds with Ji2731 and dominant exchanges single plant Genomic DNA is masterplate, and in addition to the 5th couple of primer P5, remaining primer amplifies purpose band, wherein P10 and P15 two The length of PCR product is more than using reference gene group BTx623 as the product of masterplate.And E-tian and recessive exchange single plant, from P5 PCR products are not obtained to P15, including the sequence of Dry gene the first two exons and introne about 3.6-kb, thus it is speculated that herein Sequence variations or loss occur for segment, and Dry gene functions is caused to be lost.(b) nearby genome sequence becomes Dry gene locis Different schematic diagram.Compared with the Dry genome sequences of no juice sorghum variety Ji2731, BTx623 reference gene groups include two big Fragment deletion, wherein 1.8-kb missing include sub-segments positioned at second of the gene, and the missing of 3.0-kb is happened at Dry bases Because of the upstream regions 2.5-kb；Compared to Ji2731, Dry constant gene segment Cs vary widely on E-tian genomes, thus it is speculated that occur about The missing (including Dry genome sequences about 3.6-kb) of 9.1-kb.(c) with Ji2731, E-tian, dominant exchange single plant (RIL37-18) and the recessive single plant (RIL41-11) that exchanges is that masterplate carries out Dry gene expression analysis, and SbActin is internal reference base Cause.

Fig. 4 is the sequence alignment of Dry full length genes cDNA.Four sequences are respectively：Using Ji2731 and BTx623 as mould Version, RACE methods clone the full-length cDNA of Dry genes；The BTx623 that the websites Phytozome are downloaded refers to transcript sequence；It is high The Dry gene transcripts sequences that fine strain of millet transcript profile MOROKOSHI databases are downloaded.Blue underscore represents UTR region sequence, orange Color underscore represents the gene coding regions Dry DNA sequence dna.Colored rectangles represent initiation codon and terminator codon.

Fig. 5 is the positioning of DRY protein subcellulars and situ Analysis.(a) DRY and GFP fusion proteins are thin in epidermal tobacco Born of the same parents carry out Subcellular Localization, left：Green fluorescent protein, it is right：Light field.Positive control p35S：：GFP, 40 μm of scale p35S：：10 μm of the scale of Dry-GFP figures.(b) Dry genes Sorghum Stalk situ Analysis, it is left：Antisense probe；It is right：Justice Probe (control).10 μm of scale.

Fig. 6 is transcriptional activity determination and the self-activation activity interval analysis of DRY albumen.Trp：Tryptophan； His：Group ammonia Acid；Ade：Adenine；X(X-α-Gal)：The chromogenic substrate of yeast galactosidase (MEL1)；NAM(No Apical Meristem)：The 11 to 156th amino acids of DRY albumen, including five subdomains；1-304：Including DRY albumen the 1st arrives 304 amino acids；1-268：Including the 1 to 268th amino acids of DRY albumen；1-239：Including the 1 to 239th ammonia of DRY albumen Base acid；1-235：Including the 1 to 235th amino acids of DRY albumen；1-226：Including the 1 to 226th amino acids of DRY albumen；1- 209：Including the 1 to 209th amino acids of DRY albumen；1-156：Including the 1 to 156th amino acids of DRY albumen；157-304：Packet The 157 to 304th amino acids of albumen containing DRY；252-304：Including the 252 to 304th amino acids of DRY albumen；260-304：Packet The 260 to 304th amino acids of albumen containing DRY；283-304：Including the 283 to 304th amino acids of DRY albumen.

Fig. 7 is jowar maturity period different tissues position Dry expression conditions.(a) Dry genes jowar Ji2731 not With the expression of tissue site.(b) semidefinite of the Dry genes at jowar Ji2731 and E-tian maturity period different tissues position Measure expression pattern analysis.LF, blade；LV, vein；SP, stem marrow；ESS, stem skin；ESI, internode stem skin；IMP, internode stem marrow； PA, young fringe；RT, root.Relative expression quantity：If the Dry gene expression amounts of blade are 1, Dry gene expressions in remaining different tissues It is compared with it.Three biology repeat, and significance,statistical is indicated with letter.

Fig. 8 is the Dry gene quantification PCR relative expression analysis of jowar Ji2731 heading stage stalk difference internodes.Opposite table Up to amount：It is 1 not have stalk first segment (bottom) Dry gene relative expression quantities, and Dry gene expressions of remaining difference section are with first Section is compared.Three biology repeat, and significance,statistical is indicated with letter.

Fig. 9 is Dry gene transgenic complementation tests.(a) Dry expression vectors are built.PC series be Dry genes from Body promoter and its code area；PG series is the genome sequence of corn Ubi promoters and Dry genes.Empty carrier is pCAMBIA3300.Sorghum transgenic acceptor is sugar grass Keller, the material and E-tian Dry genes having the same Missing.Riddled basins are barstar.(b) sorghum transgenosis T₁It is aqueous for plant phenotype observation, Molecular Detection and stalk It is fixed to measure.

Figure 10 is stalk without juice sorghum Ji2731 and holds Dry genome sequences comparison in juice sorghum E-tian and Keller Analysis.Blue rectangle represents NAM structural domains, and underlined in red represents exon region sequence.

Figure 11 is sorghum T₂It is investigated and analysed for transfer-gen plant with Keller economical characters are compareed, bar weight (a), plant height (b), stem thickness (c), fringe weight (d), spike length (e) and fringe handle grow (f).

Figure 12 is sorghum transgenic line and control material Sorghum Stalk marrow cell micro observation.Sorghum Stalk marrow Parenchymal tissue is observed, Keller (a) and PC4 (b).Vascular bundle microexamination, Keller (c) and PC4 (f).Vascular bundle conduit week Enclose parenchyma cell microexamination (corresponding with the cross section that white edge includes), Keller (d) and PC4 (g).Cell wall thickness is micro- It observes (corresponding with the cross section that white edge includes), Keller (e) and PC4 (h).(i) cell wall thickness of surrounding catheter parenchyma cell Degree measures.(j and k) transgenosis and the stalk proximate analysis for compareing sorghum.(j) content of cellulose measures；(k) xylan (half fiber The plain main constituents of dimension) assay.AIR, alcohol-insoluble substances.Bilateral t, which is examined, determines statistical significance, * P < 0.05, * * P < 0.01.

Figure 13 analyzes for Dry gene haplotypes.Stalk is without juice sorghum two kinds of haplotypes Ji2731-type and 2030- type；There is juice sorghum haplotype that Dry gene functions is caused to disable, respectively the Dry gene delections of E-tian-like haplotypes, There are one bases Gs to be inserted into for 2038-type haplotypes 1644, leads to frameshit, 2021-type haplotypes are in gene conserved structure Domain lacks 12 bases, and there are one base mutations at 114 of first exon for 80-type haplotypes, and coding is caused to shift to an earlier date It terminates.

Figure 14 be Dry gene translations amino acid sequence with its in sorghum, rice, corn, millet, broomcorn millet, two fringe short handles The phylogenetic analysis of ortholog translation product in grass, switchgrass and green bristlegrass.Clustal W carry out multiple sequence ratio It is right, the amino acid sequence of ortho position phase connection (Neighbor-Joining) in 6.0 softwares of MEGA to the homologous gene of Dry genes Row structure chadogram.

Specific implementation mode

Embodiment below facilitates a better understanding of the present invention, but does not limit the application range of the present invention.Following implementations All technical and scientific terms in example are unless otherwise specified that one skilled in the art of the present invention are usually managed The identical meanings of solution.Unless there are indicating on the contrary, technology that is used herein or referring to is those of ordinary skill in the art's public affairs The standard technique recognized.The test material is the general test material in field of the present invention such as without especially indicating.Following implementations Test reagent is unless otherwise specified to be commercially available from routine biochemistry reagent shop used in example.Material, method and Embodiment is sincerely used as to illustrate, rather than limits.

The present invention also holds juice including the use of sequences control plant cane described in sequence table, i.e., is provided using the present invention Gene order in genome, and/or transcript profile, and/or protein group level influence other plants identical or homologous gene Function regulate and control the purpose that cane holds juice.For example, following methods but being not limited to following methods：Pass through the change of native sequences It is different to lead to the forfeiture of gene expression inhibition or protein function, the antisense sequences by being transferred to the gene into plant or draw Enter hairpin structure or by the gene with other sequences (DNA or RNA) be combined generate the new DNA with functional activity or RNA chains, to influence or change the function of plant gene.Or other influence canes well known by persons skilled in the art that can be used for are held Any one of technical method of juice technical method.

The present invention includes sorghum Dry genes, and dominant allele holds juice to control Sorghum Stalk has crucial make With the Recessive alleles of afunction can cause moisture in stalk to increase.The gene is located at No. 6 chromosomes of sorghum, Its gene specific location is as shown in Figure 2.

The gene order and its homologous sequence can be obtained from following plant, including but not limited to sorghum (Sorghum Bicolor), corn (Zea mays), broomcorn millet (Panicum hallii), switchgrass (Panicum virgatum), green bristlegrass (Setaria viridis), millet (Setaria italica), false bromegrass (Brachypodium distachyon) and rice (Oryza sativa).Preparation method includes but not limited to：Using blastx, blastn or pass through ammonia by Dry gene orders Base acid sequence utilizes blastp from the genomic sequence data library of other plants, and/or cDNA sequence database, and/or albumen It is transferred in matter sequence library；Using the DNA or cDNA of sorghum Dry genes or RNA sequence as reference sequences design primer, directly It is directly obtained using the method for PCR from the genomic DNA or cDNA or RNA of other plants；With the gene order of sorghum Dry Design probe, using nucleic acid hybridize method detached from genomic library the DNA containing Homologous gene sequences or cDNA or RNA segments.

" Dry DNA homologs sequence " refers to after carrying out tblastn comparative analysis with the amino acid of SEQ ID NO.3, Identities is greater than or equal to 35%, Positives and is greater than or equal to 50%, and identification region is located at SEQ ID NO.3's 11 to 156 amino acids are the DNA sequences of the plant gene in conservative NAC structural domains.When carrying out blastx, all parameters In accordance with http:Default setting shown in //blast.ncbi.nlm.nih.gov/ carries out.Hereafter by illustrating and elaboration carries More detailed description is supplied, but this is not intended to limit the scope of the present invention.

1. cane of embodiment holds the identification of juice

Divide from purposes, sorghum can be divided into grain sorghum, sugar grass, Forage Sorghum and broom corn.With grain sorghum It compares, a large amount of moisture (Fig. 1 .a) is contained in sweet sorghum stalk.We hold juice by following two modes to sorghum cane Property has carried out identification：First, squeezing sorghum cane internode with pliers, if there is juice oozes out, then judge that sorghum cane holds juice, Otherwise it is determined as no juice；Second is that weighing the weight of the front and back sorghum cane of drying respectively in the maturity period, its juice containing amount is calculated.Research It was found that after blooming, the moisture rapid decrease in sorghum variety Ji2731 stalks, and sorghum variety E-tian stalks In moisture be maintained at higher level.(Fig. 1 .b).The middle part internode for taking sorghum variety Ji2731 and E-tian, through penta 2 Aldehyde and osmic acid carry out cytological observation after fixing.It is enriched it was found that the parenchyma cell of Ji2731 is more full, and E-tian Parenchyma cell is extremely irregular (Fig. 1 .d to g).Vessel cell rule in the vascular tissue of Ji2731 simultaneously, conduit week The cell wall for enclosing cell is thicker, and conduit is irregular in the vascular tissue of E-tian, and the cell wall of surrounding catheter cell is thinning (Fig. 1 .h to m).Cell wall thickness measurement result shows that the cell wall thickness of E-tian is significantly less than Ji2731 (Fig. 1 .n).It is right The cell wall constituent of Ji2731 and E-tian carries out chemical analysis, finds compared with Ji2731, the content of cellulose in E-tian It is significantly higher than Ji2731, and the content of xylan is substantially less than Ji2731 (Fig. 1 .o, p).

Embodiment 2.Dry gene finely positionings

Cane holds juice genetic analysis, constructs 4 cross combinations altogether, and respectively Ji2731 × E-tian, E-tian × Ji2731, Keller × Ji2731 and BTx623 × Ji2731.The phenotype of wherein F1 generation stalk is identical as Ji2731, shows as Phenotype (Fig. 1 .a) of the stalk without juice.F₂The size of group is respectively 214,213,114 and 221 single plants, accordingly without juice list Strain with to hold the ratio of juice single plant be respectively 150: 64,150: 63,84: 30,156: 65, different sexes show no juice single plant With the segregation ratio (table 1) held juice single plant and meet 3: 1.

1. stalk stalk of table holds the segregation ratio of juice

When carrying out the assignment of genes gene mapping, the hybrid Population used is E-tian × Ji2731, and filial generation is in water content On have larger difference (Fig. 1 .c).2-3 is chosen on every chromosome of sorghum, and PCR amplification and gel electricity are carried out to molecular labeling Swimming.As a result it shows：Two molecular labeling M1 (SM06069) on No. 6 chromosomes and M2 (6-40-2), show as The number of individuals of Ji2731 genotype shows they and Dry genes close linkage (Fig. 2 .a) more than 90%.It is passed on using single plant Mode group expand it is numerous, finally utilize 3343 single plants, the candidate section of Dry genes is navigated into molecular labeling M10 Between (6-51797) and M11 (6-51840), section size is 42kb (Fig. 2 .b).It is transferred from the phytozome of website and takes survey The genome sequence of sequence kind BTx623, discovery share 4 functional genes (Fig. 2 .c) in the sections 42Kb.

The Cloned culturing of embodiment 3.Dry genes

Based on sorghum BTx623 Genomic sequence informations, 16 pairs of primer pairs of design two hybrid strains Ji2731 and E- The gene is expanded in tian and dominant exchange single plant 37-18 and recessive exchange single plant 41-11.Wherein in Ji2731 and aobvious Sexual intercourse is changed in single plant 37-18, remove the 5th couple of primer P5 without result outside, amplification the coding region sequence for including Dry genes is obtained And upstream 7-kb, to the genome sequence in the downstream regions 2-kb, the length that wherein primer P10 and P15 corresponds to PCR product is more than Using reference gene group BTx623 as the product length of masterplate, and in E-tian and recessive exchange single plant 41-11, from P5 to P15 It does not obtain PCR product, the band (Fig. 3 .a) for meeting purpose is only obtained at primer P16.Sequence analysis is found in Ji2731 In full length gene 4155bp (SEQ ID NO.1), in contrast, sorghum reference gene group BTx623 include two large fragments Missing, wherein 1.8-kb missings include sub-segments positioned at the gene, and the missing of 3.0-kb is happened at Dry upstream region of gene 2.5- The regions kb (Fig. 3 .b).Sequence carries out blastn analyses, shows that it is single copy gene in sorghum genome.

Take two hybrid strain Ji2731 and E-tian and dominant exchange single plant 37-18 and recessive exchange single plant 41-11 yellow Change seedling, preserved with liquid nitrogen flash freezer, semi-quantitative analysis is carried out for extracting total serum IgE.SbActin genes draw as reference gene Object CDSE3_F (5 '-AGCCATGGGAGCTTCCAGAC-3 ') and CDSE3_R：(5 '-ACTGCGATGCGCCGGCAAA-3 '), For detecting Dry gene expressions, primer SbActin：Forward primer Actin_F (5 '-ACATTGCCCTGGACTACGAC-3 ') With reverse primer Actin_R (5 '-AATGAAGGATGGCTGGAAGA-3 '), the expression for detecting SbActin genes.As a result Display Dry has expression in Ji2731 and dominant exchange single plant 37-18, and without table in E-tian and recessive exchange single plant 41-11 Up to (Fig. 3 .c).

Using the RNA of Ji2731 and BTx623 as template, by 3 ' RACE and 5 ' RACE methods, expanded in two parents Dry full length genes cDNA sequence (SEQ ID NO.2).Wherein 3 ' RACE pass through two-wheeled PCR, and the primer of first round PCR is 3P1 (5 '-TGATGAACGGAGCGATGT-3 ') and B26 (5 '-GACTCTAGACGACATCGA (T) 18-3 '), the second wheel PCR processes The primer of middle amplification Ji2731 is 3P2 (5 '-TCTACGACGACAGTGGCT-3 ') and B25 (5 '- GACTCTAGACGACATCGA-3 '), expand BTx623 primer be 3P2-B (5 '-CCGACGATGATGGGGCTA-3 ') and B25(5′-GACTCTAGACGACATCGA-3′)；5 ' RACE be according to 5 ' full RACE kit of kit (TaKaRa, What experimental program Japan) carried out, two special primers be respectively GSP1 (5 '-CCACATCGCTCCGTTCAT-3 ') and GSP2(5′- TGTCTTGCTCGCTGTCTTTC-3′).Sequence alignment finds that Dry genes exist in Ji2731 and BTx623 The difference of nucleotide site, the 114th nucleotide of Dry genes causes to compile there are a base mutation (C-T) in BTx623 Code terminates (Fig. 4) in advance.And about the annotation of Dry gene C DS sequences, there are mistakes in reference gene group BTx623, by Partial sequence annotation in one exon is intron sequences.

By carrying out mock translation to cDNA sequence, which contains albumen (the SEQ ID of 304 amino acid NO.3), the protein sequence and GenBank SwissProt Protein Data Banks are subjected to blastp analyses, show that the albumen contains There are one NAC conserved domains.

Pass through genome sequence (SEQ ID NO.1) and cDNA sequence (SEQ of the Dry genes in sorghum variety Ji2731 ID NO.2) comparison analysis, find the following structural features of the gene：Contain 3 exons (Exon) and 2 intrones (Intron), wherein being first exon from the nucleotide of 5 ' end 1-169,170-1571 nucleotide are first A introne, 1572-1837 nucleotide are second exon, and 1838-3678 nucleotide are second and include Son, 3679-4155 nucleotide are third exon.

The albumen of embodiment 4.DRY codings is one transcription factor

The CDS sequences of Dry are connected to carrier pCAMBIA1205, construction of expression vector 35S：：Dry-GFP.Inject cigarette It is found after grass, DRY albumen has expression (Fig. 5 .a) only in nucleus.The CDS sequences of Dry are connected to carrier pGBKT7, Transformed yeast strains A H109, discovery can activate the expression of downstream reporter gene, and be found by the sequence analysis of some, The active region of DRY is located at the regions 236-259aa.The above experiment illustrates that the albumen of DRY codings is one transcription factor (figure 6)。

The transcriptional level of embodiment 5.Dry is analyzed

Take blade, vein, stem skin, marrow, internode skin, internode marrow, young fringe and the root of maturity period Ji2731 and E-tian, warp Liquid nitrogen flash freezer preserves, for extracting total serum IgE；After peeling stem skin off, marrow is protected with liquid nitrogen flash freezer for the internode for taking heading stage Ji2731 It deposits, for extracting total serum IgE.Above-mentioned sample standard deviation takes real-time quantitative PCR (Real-time PCR, RT-PCR) to carry out Dry genes Expression quantity detection.QRT-PCR is using SbActin genes as internal reference reporter gene, primer qPCR_F (5 '- GCTTCAGGGACCGCAAGT-3 ') and qPCR_R (5 '-GCTGTCTTTCCGCTTCTGG-3 ') be used for qRT-PCR reaction in Detect Dry gene expressions, primer Actin_F (5 '-ACATTGCCCTGGACTACGAC-3 ') and Actin_R (5 '- AATGAAGGATGGCTGGAAGA-3 ') it is used to detect the expression of SbActin genes.The Dry genes of each sample in qRT-PCR PCR reactions with SbActin genes are respectively provided with 3 independent repeated trials.

QRT-PCR is shown in blade, vein, stem skin, marrow, internode skin, internode marrow, young fringe and the root of Ji2731 and examines The expression of Dry genes is measured, and has higher expression quantity (Fig. 7) in stem skin and marrow；In the above-mentioned tissue of E-tian not Detect the expression of Dry genes.Detect the expression of Dry genes in the different internodes of Ji2731, and water content compared with The expression quantity of the low internode gene is higher.This also implies Dry gene expression regulation stalk change of moisture content (Fig. 8).

The tender internode of children for taking the after planting Ji2731 and E-tian in January, in situ hybridization is carried out after fixer FAA is fixed Experiment.The special primer used when synthesising probing needle is InSitu-F (5 '-GACAGCGAGCAAGACAACG-3 ') and InSitu-R (5′-CAGGCCACCAATACCGAG -3′).Chromogenic reaction shows Dry mainly in the parenchymal tissue and conduit of the internode of Ji2731 In have expression, and in the above-mentioned position of E-tian without expression (Fig. 5 .b).

The experiment that has complementary functions of embodiment 6.Dry

Using carrier pCAMBIA3300 as skeleton, its code area sequence driven with Dry gene own promoters is built respectively It arranges and with the carrier (Fig. 9 .a) of the genome sequence of the Dry genes of corn Ubi promoters driving.Sorghum transgene receptor material For material to hold juice sorghum variety Keller, the material is identical as E-tian genotype, shows as the missing (Figure 10) of Dry genes. Obtained transgenic line is identified, it is found that the water content of transgenic line reduces, Molecular Detection is shown in transgenosis material There are the expression of Dry genes (Fig. 9 .b) in material.Field experiment is shown, in addition to water content, transgenic line is in bar weight, plant height, Stem thickness, fringe weight, spike length and fringe handle grow (Figure 11 .a to f) identical as Keller.Cytological observation is shown, compared with Keller, is turned The parenchyma cell of genetic material is full to enrich (Figure 12 .a, b).Conduit rule, the cell of surrounding catheter cell in vascular tissue Wall is thicker (Figure 12 .c to h).Cell wall thickness measurement result shows that the cell wall thickness of transgenic line is noticeably greater than Keller (Figure 12 .i).Cell wall constituent measures display, and the content of cellulose in transgenic line cell wall is less than Keller, And the content of xylan is higher than keller (Figure 12 .j, k).

The natural variation of embodiment 7.Dry genes is analyzed

Primer P14, P15 and P16 can be used for the specific clone Dry full length genes in different sorghum strains.Three primers Combination can expand -17~+1305 ,+1214~+3958 ,+3715~+4832 segments of the gene respectively, by sequencing to expanding The gene order of increasing is verified.All PCR amplifications use KOD-Plus-Ne o (TOYOBO, Japan), and according to product The reaction system and condition of explanation carry out PCR amplification in 96 Well Thermal Cycler of ABI Veriti.PCR product is sent It is sequenced toward Beijing Sheng Gong bio-engineering corporations.It being found through sequence alignment, Dry genes include mainly 6 kinds of haplotypes, wherein There are two types of haplotype Ji2731-type and 2030-type without juice sorghum for stalk；There is juice sorghum haplotype to lead to Dry gene functions Disability, wherein in the Dry gene delections of E-tian-type haplotypes, there are one bases at 1644 for 2038-type haplotypes G is inserted into, and leads to frameshit；2021-type haplotypes lack 12 bases in gene conserved domain；80-type haplotypes are One exons 1,14 base mutations cause coding to terminate (Figure 13) in advance.

Homology of the embodiment 8.Dry genes in corn, broomcorn millet, switchgrass, green bristlegrass, millet, false bromegrass and rice point Analysis

Using the protein sequence of Dry genes, by blastp obtain the gene corn, switchgrass, broomcorn millet, millet, Ortholog in green bristlegrass, false bromegrass and rice.In corn two homologous genes be GRMZM2G043813 and GRMZM2G081930, two homologous genes are Pavir.Ga01251 and Pavir.Gb00655 in switchgrass, homologous base in broomcorn millet Because of Pahal.G01069, homologous gene is Seita.7G166500 in millet, and homologous gene is in green bristlegrass Sevir.7G175600, homologous gene is Bradi5g15587 in false bromegrass, and homologous gene is LOC_Os04g43560 in rice (Figure 14).

Although the specific implementation mode of the present invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change the guarantor in the present invention Within the scope of shield.The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims (10)

1. a kind of regulation and control plant stem holds the new gene Dry of juice, it is characterised in that：Be it is following 1) or 2) or 3) or 4) or 5) DNA molecular：

1) DNA molecular shown in SEQ ID NO.1；

2) DNA molecular shown in SEQ ID NO.2；

3) on the bases SEQ ID NO.1, by one to several bases replace and or one to several bases insertion and or Missing and large fragment nucleotide sequence be inserted into missing displacement inversion formed can regulate and control cane hold juice DNA divide Son；

4) 0.1 × SSPE or 0.1 × SSC, 0.1%w/v SDS solution in, hybridize and wash film, Neng Gouyu under the conditions of 65 DEG C The DNA molecular of SEQ ID NO.2 hybridizes and regulates and controls the DNA molecular that cane holds the GAP-associated protein GAP of juice；

5) with the DNA molecular of SEQ ID NO.2 with 70% or more homology and coded plant moisture regulation GAP-associated protein GAP DNA molecular.

2. the protein of gene code according to claim 1, which is characterized in that be selected from it is following 1) or 2) described in albumen Matter：

1) protein that amino acid sequence shown in SEQ ID NO.3 forms.

2) SEQ ID NO.3 are passed through into the substitution of one or several amino acid residues and/or lacks and ors add and there are regulation and control Cane holds the protein of the related activity of juice.

3. the expression cassette, recombinant vector, transgenic cell line containing gene described in claim 1 or recombinant bacterium.

4. containing gene order described in claim 1, it is inserted into, and/or lacks, and/or replace the controllable of several nucleotide Cane holds the DNA molecular of juice.

5. the gene of the claims 1 or 2 and/protein are in the purposes for transgene improvement crop.

6. the purposes described in claim 5, wherein the gene holds juice to obtain good transgenosis work for regulating and controlling cane Object.

7. the purposes described in claim 5-6, wherein the improvement includes that can be used for bio-ethanol, syrup and ensilage Quality-improving.

8. the purposes described in claim 5-6, wherein the crop is self-pollination or cross pollinated plant；Further, institute It includes but not limited to sorghum, corn, wheat, false bromegrass or rice to state crop.

9. a kind of method can be used for obtaining ortholog of the gene in other plants described in claim 1, the method For：1) use protein sequence described in claim 2 in Genbank RiboaptDBs http：// Tblastn search is carried out in www.ncbi.nlm.nih.gov/genbank/；2) Identities be greater than or equal to 35%, Positives is greater than or equal to 50%, and it is the NAC guarded that identification region, which is located at 11 to 156 amino acids of SEQ ID NO.3, The DNA sequence dna of plant gene in structural domain is the gene with gene ortholog described in claim 1.

10. based on gene described in claim 1 in corn (Zea mays), broomcorn millet (Panicum hallii), switchgrass (Panicum virgatum), green bristlegrass (Setaria viridis), millet (Setaria italica), false bromegrass The amino acid sequence of ortholog in (Brachypodium distachyon) and rice (Oryza sativa), specifically As shown in figure 14.