CN106967702A - A kind of cellulase and its encoding gene and application - Google Patents

Abstract

The invention discloses a kind of cellulase and its encoding gene and application.The amino acid residue sequence of the cellulase is such as shown in (a) or (b)：(a) such as SEQ ID No：Amino acid residue sequence shown in 1；(b) to such as SEQ ID No：Amino acid residue sequence shown in 1 is by 1 or the substitution of several amino acid residues and/or missing and/or addition and the amino acid sequence with regulating and controlling effect that ftractureed to plant silique.Cellulose enzyme gene of the present invention is mutated or can significantly postpone the cracking time of silique after being suppressed expression, the cracking rate of plant silique is reduced simultaneously, cause the seed underproduction because environmental factor causes respective crop silique to ftracture in advance so as to reduce and cause the loss of land pollution, and then ensure the high production of crop seed, with higher actual application value, have a extensive future.

Description

A kind of cellulase and its encoding gene and application

Technical field

The invention belongs to gene engineering technology field, more particularly to a kind of cellulase and its encoding gene and application.

Background technology

In the life process of plant, very important role is served as in the separation of cell：Sprouted from seed, to moulding The form of plant, propagates pollen from anther dehiscence, is distributed to cracking of fruit seed, all with cell on microcosmic and macroscopic level " separation " is closely related.The fruit of numerous species is (for example：Silique, capsule, Follicle radish fruit, pod etc.) in the later stage rank of its development Section, can all occur the differentiation of the cell of specific type and a series of molecules and biochemical event being closely related with it, finally make Mellow fruit cracking, and discharge seed, so as to be conducive to expanding the growth scope of Progeny plants, be flourishing race and The adaptability for improving plant provides condition and guarantee.

The cracking of fruit, along with the propagation of seed, is the important way that plant completes the continuity of life and the procreation of race Footpath.However, in agricultural production, crops cause Fruit pod premature rupture often to directly affect the receipts of crop because of climate change Into causing a large amount of wastes.Such as annual production of oil crops colea, the loss caused due to cracking of fruit is every year on average Up to 20%, (Child R.D.et al., 1998, Ethylene up to 50% are lost in adverse weather conditions biosynthesis in oilseed rape pods in relation to pod shatter.Journal of Experimental Botany 49:829–838).Meanwhile, cracking of fruit can also limit the further hair of oil crop seeds Educate, influence oil content (Jenkins the E.et al., 1999, Dehiscence-related expression of an of seed Arabidopsis thaliana gene encoding a polygalacturonase in transgenic plants of Brassica napus.Plant,Cell and Environment 22:159-167.).In addition, being dissipated because of cracking of fruit The seed of hair can also pollute (Spence J.et al., 1996, ' Pod shatter ' in other crops and environment Arabidopsis thaliana,Brassica napus and B.juncea.Journal of Microscope 181: 195-203).As can be seen here, research cracking of fruit has directly and far-reaching influence to agricultural production.

Arabidopsis is the conventional model plant of botany research, equal with traditional oil plant crop oil dish etc., the development of its Fruit pod There is similar design feature to cracking to traditional oil crops.Have growth cycle short (2 months compared with traditional oil crops A life cycle can be completed), all sequencing is finished (gene order understands) gene, and various correlative study data are very rich The advantages such as richness, the method and thinking of the improvement of many oil crops kinds all derive from the correlative study of arabidopsis.Such as regulate and control to intend One transcription factor FRUITFUL (FUL) of southern mustard Fruit pod cracking, there are some researches show replacing coding by using 35S promoter should The promoter of transcription factor gene, makes the gene ectopic expression to cause (the Ferr á ndiz that do not ftractureed after the Fruit pod of arabidopsis is ripe C.et al.,2000,Redundant regulation of meristem identity and plant architecture by FRUITFULL,APETALA1,and CAULIFLOWER.Development 127:725-734)。 And the later stage, in the research of colea, scientist obtains the FUL homologous genes of colea by the method for homologous comparison, And the gene ectopic expression is made by identical method, do not ftractureed strain (Qstergaard so as to filter out colea fruit L.et al.,2005,Pod shatter-resistant Brassica fruit produced by ectopic expression of the FRUITFULL gene.Plant Biotechnology Journal 4:45-51).Thus may be used See the mechanism of the Fruit pod cracking of research arabidopsis, strong instruct and strong can be provided for other plant (particularly oil crops) research Big theoretical foundation.

Cellulase, hemicellulase and pectase be degradation of cell wall main three kinds of enzymes (Patterson S., 2001,Cutting loose,Abscission and dehiscence in Arabidopsis.Plant Physiology126:494-500；Roberts J.A.et al.,2000,Cell separation processes in plants:models,mechanisms and manipulation.Annals of Botany 86:223-235).And plant The process for coming off and separating of organ is usually associated with the cell wall degradation of dehiscence zone cell.There are some researches show declined in Fruit pod maturation The enhancing in the cellulase activity of dehiscence zone cell is usually associated with during old and has fairly obvious specificity (Meakin And Roberts, 1990, Dehiscence of fruit in oilseed rape (Brassica napus L.) .I.Anatomy of pod dehiscence.Journal of Experimental Botany 41:1003-1011)；Really Although the activity change of the adjoint Fruit pod ripening and senscence of glue enzyme is less and without specificity, the gene of mutation coding pectase (ADPG1 and ADPG2) but causes Fruit pod can not ftracture (Ogawa M.et al., 2009, RABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE1(ADPG1),ADPG2,and QUARTET2Are Polygalacturonases Required for Cell Separation during Reproductive Development in Arabidopsis.Plant cell 21:216-233).But participate in the cellulose enzyme gene and hemicellulase of Fruit pod cracking Gene is still unknown, identifies the related cellulase of arabidopsis Fruit pod cracking and hemicellulase ftractureed abundant to Fruit pod The understanding of cell wall degradation process in journey, and the have an opportunity gene and phenotype that find new influence cracking of fruit, and then to rape The seed selection of new varieties provides new research direction and thinking, so as to increase the yield of rape.With being obtained by regulative transcription factor Indehiscent napus lines compare, by regulate and control downstream enzymes obtain the strain that do not ftracture more have with production application value, Because transcription factor is that the formation in whole Fruit pod cracking area is influenceed by regulating and controlling the expression of multiple genes, thus regulatory transcription factor Strain produced by son typically all can cause fruit not split completely, can be added on the contrary because of increase workload in production This, loses more than gain compared with the loss caused by the underproduction that crack band comes.And regulate and control the product produced by downstream cellular wall hydrolase System only can influence an expression conditions, whole Fruit pod cracking area can't be formed and cause tremendous influence, therefore typically The postponement and cracking rate reduction of cracking of fruit are only resulted in, fruit can't be caused not split completely.It so can both reduce cracking The loss caused, will not increase the cost of production again, therefore with more practical application meaning and prospect.

The content of the invention

The primary and foremost purpose of the present invention is to overcome the shortcoming and deficiency of prior art, and there is provided a kind of cellulase, the fiber Plain enzyme has regulating and controlling effect to plant silique cracking.

Another object of the present invention is to provide the encoding gene of the cellulase.

A further object of the present invention is the application for providing the cellulase.

The purpose of the present invention is achieved through the following technical solutions：A kind of cellulase, entitled CEL6, from arabidopsis (Arabidopsis thaliana), its amino acid residue sequence is such as shown in (a) or (b)：

(a) such as SEQ ID No：Amino acid residue sequence shown in 1；

(b) to such as SEQ ID No：Amino acid residue sequence shown in 1 is by 1 or the substitution of several amino acid residues And/or missing and/or addition and the amino acid sequence with regulating and controlling effect that ftractureed to plant silique.

Wherein, the SEQ ID No in sequence table:1 is made up of 497 amino acid residues, and its molecular weight is 55kDa.

Cellulase in above-mentioned (a) and (b) can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological table Reach；(b) CEL6 encoding gene can be by by sequence SEQ ID No in:Lacked in DNA sequence dna in 1 one or several The codon of amino acid residue, and/or the missense mutation of one or several base-pairs of progress are obtained.

The encoding gene of described cellulase, its nucleotide sequence is such as shown in (a), (b), (c) or (d)：

(a) such as SEQ ID No：Polynucleotides shown in 2；

(b) coding such as SEQ ID No：The polynucleotides of amino acid shown in 1；

(c) with SEQ ID No：Polynucleotides shown in 2 have more than 80% homology and plant silique are ftractureed and have There is the nucleotide sequence of regulating and controlling effect；

(d) under high high stringency conditions with SEQ ID No：The nucleotide sequence of the 2 DNA sequence dna hybridization limited.

Wherein, the SEQ ID No in sequence table：2 by 3282 base compositions, and its coding region is 82-504,1457- Five regions of 1756,1927-2088,2310-2585,2690-3022.

Above-mentioned high high stringency conditions can be in 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS solution, it is miscellaneous at 65 DEG C Hand over and wash film.

Recombinant vector, recombinant microorganism, transgenic cell line or expression cassette containing any of the above-described encoding gene Belong to protection scope of the present invention.

The recombinant vector is that any of the above-described encoding gene is inserted into the polyclonal of carrier pCAMBIA1305.1 that set out The recombinant expression carrier that site is obtained.

Described recombinant microorganism is that the recombinant vector is converted into the recombinant microorganism that bacterium obtains.

Application of the described cellulase in regulation and control plant silique cracking gene.

The plant includes monocotyledon and dicotyledon, preferably arabidopsis or rape variety.

Application of the described cellulase in regulation and control plant silique cracking gene, is according to described cellulose enzyme gene Sequence, disturbs (RNA intrference, RNAi), antisense RNA (anti-sense RNA), overexpression negative dominant using RNA (dominant negative, DN), crispr/cas9 gene editing technologies make normal plants cellulase encoding gene The function of CEL6 homologous gene is lost or expression quantity declines, so as to realize that postponement plant silique cracking and reduction plant silique are opened Split rate.

The present invention has the following advantages and effect relative to prior art：

1st, (1) present invention analyzes cellulose enzyme gene contained by current database by existing reverse genetics theoretical foundation Relevant information data, illustrate cellulose enzyme gene participate in regulation and control Rape pod development and cracking mechanism, and be subject to qPCR measure The expression quantity change of arabidopsis silique different development stage, the specific participation Rape pod development and the cellulase of dehiscence process obtained is compiled Code gene C EL6；(2) present invention is demonstrated by mutant strain silique dehiscence zone cell Ultrastructure analysis and correlation statistical analysis The function of the gene there is provided cellulose enzyme gene there is important application value, be according to the gene sequence information utilize RNA interference (RNAi), antisense (RNA anti-sencse RNA), be overexpressed bear dominant (dominant negative, DN) or Crispr/cas9 gene editing technologies make normal plants (such as arabidopsis or rape variety) cellulase encoding gene CEL6 The function of homologous gene is lost or expression quantity declines, and plant silique cracking rate is ftractureed and reduce so as to realize and postpone plant silique Method.

2nd, the cellulase in the present invention is cultivating the application during plant silique delays cracking, and described plant silique can be with It is indehiscent silique strain.

3rd, gene of the invention is mutated or can significantly postpone the cracking time of silique after being suppressed expression, is planted while reducing The cracking rate of object angle fruit, so that reduce causes the seed underproduction and cause because environmental factor causes respective crop silique to ftracture in advance The loss of land pollution, and then ensure the high production of crop seed, with higher actual application value, have a extensive future.

Brief description of the drawings

Fig. 1 is the solubility curve figure in embodiment 2, wherein, figure a represents to dissolve peak, and figure b represents solubility curve.

Fig. 2 is that the cellulase gene C EL6 of embodiment 2 unites in the quantitative PCR expression of results of the Fruit pod of different development stage Meter figure, wherein, 6D, 8D, 10D, 12D represent the number of days of Post flowering；18A represents half yellow fruit pod；18B represents complete yellow Fruit pod.

Fig. 3 is the cellulase gene C EL6 of embodiment 3 gene schematic diagram and the material of two kinds of cellulase mutant strains The electrophoresis result figure of the establishment of the insertion point of material；Wherein, Col-0 represents wild type, and cel6-1 and cel6-2 represent saltant type.

Fig. 4 is the t-DNA of the cellulase gene C EL6 of embodiment 4 mutant strain cel6-1 and cel6-2 homozygote material The schematic diagram of insertion point.

Fig. 5 is the maturation of the cellulase gene C EL6 of embodiment 5 mutant strain cel6-1 and cel6-2 homozygote material The comparing result figure of the expression of CEL6 genes and wild type in Fruit pod, wherein, Col-0 represents wild type.

Fig. 6 is the system of wild type and cellulase CEL6 mutant strains Fruit pod number of days needed for from blooming to turning yellow in embodiment 6 Meter compares figure, wherein, Col-0 represents wild type, and cel6-1 and cel6-2 represent saltant type.

Fig. 7 is wild type and cellulase mutant strain cracking rate statistical results chart in embodiment 7, wherein, Col-0 represents wild Raw type, cel6-1 and cel6-2 represent saltant type, and 18 phases represented arabidopsis yellow fruit pod, and 19 phases represented fruit development to firm browning Fruit pod.

Fig. 8 be cellulase mutant strain cel6-1 and cel6-2 the homozygote material of embodiment 8 Fruit pod it is ripe, turn yellow, The microstructure variation diagram in the stages such as cracking.Wherein, Col-0 represents wild type, and cel6-1 and cel6-2 represent saltant type；18A Represent half yellow fruit pod；18B represents complete yellow Fruit pod；19A represents dry brown Fruit pod.

Embodiment

With reference to embodiment, the present invention is described in further detail, but the implementation of the present invention is not limited to this.

The clone of the cellulase CEL6 genes of embodiment 1

The acquisition of the full length gene DNA sequence dna of arabidopsis cellulase, comprises the following steps：

1. in TAIR databases (http://www.arabidopsis.org) in using cellulase (cellulase) as Keyword is scanned for, and the cDNA sequence for searching for gained gene is carried out into Blast comparisons in TAIR databases respectively, takes ratio Candidate gene (totally 39) are used as to all genes for being less than 0.01 of E-Value in result.

2. by the gene expression chip (microarray of the candidate gene of gained respectively in TAIR databases Expression) database is scanned for, and will have the gene expressed in 15 phase of flower development carpel to retain, remaining rejects (remaining 22).

3. remaining candidate gene is inputted into ATTED-II databases (http://atted.jp) middle progress correlation coexpression Gene is inquired about.Found in 300 related genes obtained by inquiry and develop the transcription factor related to cracking (such as with Fruit pod SHP1, SHP2, ALC etc.), the candidate gene that the transcription factor related with cracking can be developed to Fruit pod has coexpression result leaves, Remaining is rejected (remaining 6).

4. with indehiscent transcription factor mutant strain (Fruit pod do not ftracture transcription factor mutant ind-7) and WT (wild types Col-0) it is material, carries out whether the remaining candidate gene of quantitative PCR detection table in difference, wherein WT occurs in two kinds of materials Up to reservation of the amount than the mutation plant height that do not ftracture, remaining is rejected (remaining two).Wherein, Fruit pod does not ftracture transcription factor mutant Ind-7 is obtained by the following method：

According to document (Liljegren, S.J., Roeder, A.H., Kempin, S.A., Gremski, K.,L.,Guimil,S.,Reyes,D.K.,and Yanofsky,M.F.2004.Control of fruit patterning in Arabidopsis by INDEHISCENT.Cell.116:843-853；Wu,H.,Mori,A.,Wang, Y.X.,and Yang,M.2006.The INDEHECIENT protein regulates unequal cell division in Arabidopsis fruits.Planta.224:Fruit pod is by nothing after arabidopsis transcription factor IND mutation known to 971-979) Method ftractures.Arabidopsis IND inactivated homozygous mutant bodies are screened by following methods：From ABRC (Arabidopsis Biological Resource Center) obtain IND (AT401200) site T-DNA insertion mutant strain seed (seed number： SALK_058083), and it is named ind-7 (Alonso J.M., Stepanova A.N., Leisse T.J., Kim C.J., Chen H.,Shinn P.,Stevenson D.K.,Zimmerman J.,Barajas P.,Cheuk R.,et al.2003.Genome-wide insertional mutagenesis of Arabidopsis thaliana.Science 301:653-657.), the primer and according to needed for the Data Design that website is provided, primer sequence is as follows：

IND identifies sense primer：5’-CACCCATACAATCTTGGATCG-3’；

IND identifies anti-sense primer：5’-TGTTCTGGTTGGGTTCAAAAG-3’；

SALK_058083 inserts primer：5’-TGGTTCACGTAGTGGGCCATCG-3’；

PCR reaction systems are as follows：

PCR response procedures are：

Using above-mentioned PCR reaction systems and response procedures, with the DNA (CTAB methods) of the arabidopsis of extraction for template, use IND identifies that sense primer and IND identification anti-sense primers can amplify one in the arabidopsis DNA of wild type (Col-0) 1198bp band, and correspondence band can not be amplified in ind-7 homozygous mutation bodies.And use SALK_058083 to insert Enter primer and IND identification anti-sense primers, the band that length is respectively 756bp can be amplified in ind-7 homozygous mutation bodies, And correspondence band can not be obtained in the DNA of the arabidopsis of wild type again.So as to illustrate that we are identified by PCR, two kinds are obtained The sub- mutant of IND inactivated homozygous of different T-DNA insertion points, is named as ind7 (SALK_058083).

5. according to the related data of this gene in this database, in the design of the 5 ' of the gene order and 3 ' end sequences Pair of primers, primer sequence is as follows：

CEL6 cloned upstream primers：

5’-GGACTCTTGACCATGGTAGTTGCAAACACCGATC-3’；

CEL6 cloned downstream primers：

5’-ATTTACCCTCAGATCTACCATAGCAACCATTATATATATGTATA-3’；

The STb gene (CTAB methods) of arabidopsis is extracted, in the guiding of CEL6 cloned upstreams primer and CEL6 cloned downstream primers Under, using the STb gene of arabidopsis as template, PCR amplification arabidopsis cellulose enzyme gene full length DNA sequences, 50ul PCR reactants It is to be：STb gene template 2 μ l, Premix Taq (Ex Taq Version2.0；Takara companies) 25 μ l, CEL6 cloned upstreams draw Thing (2mM) 5 μ l, CEL6 cloned downstream primers (2mM) 5 μ l, water (ddH₂O)13μl.PCR reaction condition is：First 94 DEG C of denaturation 2 Minute；Then 94 DEG C are denatured 30 seconds, 58 DEG C of renaturation 1 minute, and 72 degrees Celsius extend 4 minutes, totally 35 circulations.After reaction terminates, 0.8% agarose gel electrophoresis detection is carried out to PCR primer, reclaims and purifies the amplified fragments that length is 3200bp, use Clotech infusion-clone technologies are cloned into pCAMBIA1305.1 carriers, are obtained containing the weight for reclaiming fragment Group plasmid, uses M13 primer pairs (M13 sense primers：5’-GAGCGGATAACAATTTCACACAGG-3’；M13 anti-sense primers： 5 '-CGCCAGGGTTTTCCCAGTCACGAC-3 ') it is sequenced, and sequencing result shows arabidopsis cellulase CEL6 genes Total length has SEQ ID No in sequence table：2 nucleotide sequence, by 3282 base compositions, by sequence alignment, with intending south The gene order is classified as cellulase family, is named as by the homology between mustard cellulase family member more than 80% Cellulase CEL6 is (referred to as：CEL6)；Its coding region be 82-504,1457-1756,1927-2088,2310-2585, This five regions of 2690-3022, protein of the coding with SEQ ID No.1 amino acid sequence in sequence table, it encodes egg It is white to be named as cellulase CEL6 (referred to as：CEL6), its molecular weight is 55kDa.

Embodiment 2：Cellulase CEL6 is in the developmental expression change of Fruit pod

Marked when wildtype Arabidopsis thaliana material (Col-0) is bloomed on anthocaulus with the rope of different colours, record pair Time when blooming answered, then according to the number of days (6D, 8D, 10D, 12D) and the color change (18A of Fruit pod of Post flowering：Half Yellow fruit pod；18B：Complete yellow Fruit pod) drawn materials, use QIAGEN companies using the material of gained as sample Plant Mini Kit extract RNA, and reverse transcription is into cDNA, makees three groups of samples parallel.Then using progress quantitative PCR detection CEL6 genes are in the expression quantity of different development stage Fruit pod, and the quantitative primer used is as follows：

CEL6 quantitative PCR sense primers：5’-TTTCCCGATGGCTTTCACCA-3’；

CEL6 quantitative PCR anti-sense primers：5’-GAGAGCCACGAGAGAGTTACG-3’；

Internal reference actin8 sense primers：5’-TAAGGTCGTGGCACCACCCG-3’；

Internal reference actin8 anti-sense primers：5’-ATCCGAGTTTGAAGAGCTACAA-3’.

Reaction system used is：

Response procedures are：

Dissolving program, obtains solubility curve (see Fig. 1).

Reacted using Bio-rad CFX96TouchTM fluorescence quantitative PCR detection systems operation quantitative PCR.As a result such as Fig. 2 It is shown, wherein, period 17 represents a stage of development of Fruit pod, refers to that departing from Fruit pod from floral organ (flower pesticide, sepal etc.) arrives The Fruit pod in Fruit pod flavescence (18 phase) this stage.As can be seen from the figure cellulose enzyme gene CEL6 Fruit pod develop and extend The expression quantity in period (6D, 8D, 10D, 12D) is higher than period (18A and 18B) expression quantity that Fruit pod aging turns yellow, and expression quantity is most High period be Post flowering 8D (my god) when Fruit pod.The result shows, the gene with Fruit pod correlation of growing, in fruit Cracking area just has begun to work when newly formed, and affects the Morphogenesis in Fruit pod cracking area.

Embodiment 3：The screening of cellulase CEL6 mutant strains

Arabidopsis CEL6 inactivated homozygous mutant bodies are screened with following methods：From ABRC (ArabidopsisBiological Resource Center) obtain two kinds of CEL6 (AT4G39010) site it is different T-DNA insertion mutant strain seed (seed is numbered：SALK_060505 and WiscDsLox485-488K15) (Alonso J.M., Stepanova A.N., Leisse T.J.,Kim C.J.,Chen H.,Shinn P.,StevensonD.K.,Zimmerman J.,Barajas P.,Cheuk R.,et al.2003.Genome-wide insertionalmutagenesis of Arabidopsis thaliana.Science 301:653-657.), the primer and according to needed for the Data Design that website is provided, primer sequence is as follows：

CEL6 identifies sense primer：5’-CGTATTATCCAAGCATAAATCGC-3’；

CEL6 identifies anti-sense primer：5’-TAGCTGTGAAACGGATTTGAGG-3’；

SALK_060505 Insert Fragment primers：5’-TGGTTCACGTAGTGGGCCATCG-3’；

WiscDsLox485-488K15 Insert Fragment primers：

5’-AACGTCCGCAATGTGTTATTAAGTTGTC-3’。

Using the DNA of arabidopsis as template (the arabidopsis DNA that embodiment 1 is extracted), use, anti-sense primer is expanded, PCR Reaction system is as follows：

PCR response procedures are：

Identified by PCR, obtain the sub- mutant of CEL6 inactivated homozygous of two kinds of different T-DNA insertion points, Cel6-1 (SALK_060505) and cel6-2 (WiscDsLox485-488K15) are respectively designated as, detailed process is：

Using above-mentioned PCR reaction systems and response procedures, identify that sense primer and CEL6 identify anti-sense primer using CEL6 620bp band can be amplified in the arabidopsis DNA of wild type (Col-0), and in cel6-1 and cel6-2 homozygotes Correspondence band (Fig. 3 A and 3B) can not be amplified in mutant.And use SALK_060505 Insert Fragments primer and CEL6 to reflect Determine anti-sense primer, WiscDsLox485-488K15 Insert Fragments primer and CEL6 identification sense primers can be respectively in cel6-1 With amplify the band that length is respectively 838bp and 488bp in cel6-2 homozygous mutation bodies, and can not wild type again plan south Correspondence band (Fig. 3 C and 3D) is obtained in the DNA of mustard.

Embodiment 4：The identification of the t-DNA insertion points of the sub- mutant of CEL6 inactivated homozygous

Anti-sense primer will be identified using SALK_060505 Insert Fragments primer and CEL6, using cel6-1 DNA as template Pcr amplification product and use WiscDsLox485-488K15 Insert Fragments primer and CEL6 identify sense primer, with cel6-2's DNA be sequenced respectively for the pcr amplification product of template (cel6-1 DNA and cel6-2 DNA by 838bp in embodiment 3 and 488bp band is reclaimed and obtained).According to the sequencing result of gained in arabidopsis database Blast with determine cel6-1 and Cel6-2 T-DNA insertion point.As a result show, cel6-1 insertion point is located at before 5 '-Utr of CEL6 genes 7 At base, belong to CEL6 promoter regions；And cel6-2 insertion point is located on first extron of CEL6 genes (see figure 4)。

Embodiment 5：Verify that the expression of CEL6 genes in the sub- mutant of CEL6 inactivated homozygous is lowered

The Post flowering Fruit pod of 12 days using cel6-1 and cel6-2 is material extraction RNA respectively, and reverse transcription is into cDNA.With CDNA is template, and Colombia's type (Col-0) is that wild type is control, consistent with embodiment 2 using quantitative primer.As a result as schemed Shown in 5, it can be seen that in two kinds of sub- mutant of CEL6 inactivated homozygous of gained the expression quantity of CEL6 genes only have it is wild 14% (cel6-1) and 6% (cel6-2) of type.

Embodiment 6：Cellulase variants Fruit pod maturation is postponed

By the cellulase CEL6 of embodiment 2 homozygous mutation body cel6-1 and cel6-2 and correspondence wild type (brother Rival hypotype) arabidopsis material flower by tying up date of the different colours rope flower that is marked that the flowers are in blossom recording every, so It can be calculated from flower to the development time needed for flavescence Fruit pod when fruit turns yellow by observing rope color afterwards, every kind of material system Count at least 50 Fruit pods.As a result as shown in fig. 6, it can be seen that wildtype Arabidopsis thaliana (Col-0) becomes from blooming Fruit pod Huangping is required to 14.25 days, and cellulase homozygous mutation body cel6-1 and cel6-2 be then respectively necessary for 17.41 days and 17.56 days, postponed 3 days or so than wild type.Illustrate that cellulase missing can cause arabidopsis fruit development to postpone.

Embodiment 7：Cellulase variants Fruit pod cracking rate declines

By the cellulase CEL6 of embodiment 2 homozygous mutation body cel6-1 and cel6-2 and correspondence wild type brother's human relations Than the arabidopsis material of hypotype (Col-0) fruit development to firm browning (19 phase) when, take the fruit corresponding with this Fruit pod Upper one complete yellow Fruit pod (18 phase) is positioned under anatomical lens and has seen whether breach.Every kind of material (cel6-1, cel6-2, Col-0) chosen from more than 6 plants of plant：The Fruit pod of 15 18 phases of every Plant statistics and 15 19 phases (count altogether 90 Above Fruit pod), (cracking rate is the percentage that fatiscent Fruit pod accounts for Fruit pod sum to the cracking rate in calculating every kind of material every kind of period Than), then every kind of material repeats (every kind of material every kind of period can obtain 6 cracking rates) with 6 plants of plants as sample, then counts Average cracking rate is obtained, the cracking rate of the different times of mutant strain and corresponding wild type data are being compared and P is used Value verifies their otherness.

As a result as shown in fig. 7, it can be seen that the cracking rate of wildtype Arabidopsis thaliana yellow fruit pod (18 phase) be 76 ± 3.6%, and the cracking rate of cellulase homozygous mutation body cel6-1 and cel6-2 yellow fruit pod (18 phase) is 10 ± 4.2% and 20 ± 4.4%；In browning Fruit pod, the cracking rate of wild type is 100%, and the cracking rate of mutant strain be 42 ± 6.5% and 44 ± 10%.The P values of mutant strain versus wild type are far below 0.05, show that mutant strain and wild type exist between cracking rate notable Significant difference.After illustrating cellulase missing arabidopsis cracking of fruit rate can be caused to decline.

Embodiment 8：Influence of the cellulase variants Fruit pod to structure in Fruit pod growth course

Take wild type (Col-0), the sub- mutant strain cel6-1 and cel6-2 of cellulase CEL6 inactivated homozygous half yellow fruit pod (18A), complete yellow Fruit pod (18B) and dry brown Fruit pod (19A) are detected in Stereo microscope.As a result it is as shown in Figure 8： The yellow period (18A) of Fruit pod half, wild type and the sub- mutant strain cel6-1 and cel6-2 of cellulase CEL6 inactivated homozygous fruit Pod is not ftractureed.In the period of Fruit pod is complete yellow (18B), the fruit lobe and replum of wild type have been separated, and Fruit pod has begun to out Split, and the sub- mutant strain cel6-1 and cel6-2 of cellulase CEL6 inactivated homozygous Fruit pod is not split.In dry brown Fruit pod (19A) In, the Fruit pod of wild type is split completely, and the sub- mutant strain cel6-1 and cel6-2 of cellulase CEL6 inactivated homozygous fruit The whole cracking area of pod is still in the state that do not ftracture.Illustrate to cause tremendous influence to the cracking of Fruit pod after cellulase mutation.

Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

SEQUENCE LISTING

<110>Agricultural University Of South China

<120>A kind of cellulase and its encoding gene and application

<130> 1

<160> 17

<170> PatentIn version 3.5

<210> 1

<211> 497

<212> CEL6

<213>Arabidopsis arabidopsis（Arabidopsis thaliana）

<400> 1

Met Lys Leu His Leu His Pro Leu Ser Leu Val Phe Phe Phe Phe Phe

1 5 10 15

Phe Phe Phe Arg Pro Thr Met Ser Ser Asn Gln His Asp Tyr Ser Asp

20 25 30

Ala Leu Ser Lys Ser Ile Leu Phe Phe Glu Gly Gln Arg Ser Gly Tyr

35 40 45

Leu Pro Asn Asp Gln Arg Met Thr Trp Arg Arg Asn Ser Gly Leu Ser

50 55 60

Asp Gly Trp Thr His Asn Ile Asp Leu Thr Gly Gly Tyr Tyr Asp Ala

65 70 75 80

Gly Asp Asn Val Lys Phe Asn Phe Pro Met Ala Phe Thr Thr Thr Met

85 90 95

Leu Ala Trp Ser Val Ile Glu Phe Gly Glu Phe Met Pro Ser Ser Glu

100 105 110

Leu Arg Asn Ser Leu Val Ala Leu Arg Trp Ser Ser Asn Tyr Leu Leu

115 120 125

Lys Ser Val Ser Gln Leu Pro Asn Arg Ile Phe Val Gln Val Gly Asp

130 135 140

Pro Ile Ala Asp His Asn Cys Trp Glu Arg Pro Glu Asp Met Asp Thr

145 150 155 160

Pro Arg Thr Ala Tyr Ala Val Asn Ala Pro Asn Pro Ala Ser Glu Val

165 170 175

Ala Gly Glu Thr Thr Ala Ala Leu Ser Ala Ala Ser Ile Ala Phe Arg

180 185 190

Ser Ser Asp Pro Gly Tyr Ser Gln Thr Leu Leu Gln Asn Ala Val Lys

195 200 205

Thr Phe Gln Phe Ala Asp Met Tyr Arg Gly Ala Tyr Ser Ser Asn Asp

210 215 220

Asp Ile Lys Asn Asp Val Cys Pro Phe Tyr Cys Asp Phe Asn Gly Phe

225 230 235 240

Gln Asp Glu Leu Leu Trp Gly Ala Ala Trp Leu Arg Lys Ala Thr Gly

245 250 255

Asp Glu Ser Tyr Leu Asn Tyr Ile Glu Ser Asn Arg Glu Pro Phe Gly

260 265 270

Ala Asn Asp Asn Val Asp Glu Phe Gly Trp Asp Asn Lys Val Gly Gly

275 280 285

Leu Asn Val Leu Val Ser Lys Glu Val Ile Glu Gly Asn Met Tyr Asn

290 295 300

Leu Glu Ala Tyr Lys Ala Ser Ala Glu Ser Phe Met Cys Ser Leu Val

305 310 315 320

Pro Glu Ser Ser Gly Pro His Val Glu Tyr Thr Ser Ala Gly Leu Leu

325 330 335

Tyr Lys Pro Gly Gly Ser Gln Leu Gln His Ala Thr Thr Ile Ser Phe

340 345 350

Leu Leu Leu Val Tyr Ala Gln Tyr Leu Ser Arg Ser Ser Leu Ser Leu

355 360 365

Asn Cys Gly Thr Leu Thr Val Pro Pro Asp Tyr Leu Arg Arg Leu Ala

370 375 380

Lys Lys Gln Val Asp Tyr Ile Leu Gly Asn Asn Pro Met Gly Leu Ser

385 390 395 400

Tyr Met Val Gly Tyr Gly Glu Arg Tyr Pro Lys Arg Ile His His Arg

405 410 415

Gly Ser Ser Leu Pro Ser Ile Val Asp His Pro Glu Ala Ile Arg Cys

420 425 430

Lys Asp Gly Ser Val Tyr Phe Asn Ser Thr Glu Pro Asn Pro Asn Val

435 440 445

Leu Ile Gly Ala Val Val Gly Gly Pro Gly Glu Asp Asp Met Tyr Asp

450 455 460

Asp Asp Arg Ser Asp Phe Arg Lys Ser Glu Pro Thr Thr Tyr Ile Asn

465 470 475 480

Ala Pro Phe Val Gly Val Leu Ala Tyr Phe Ala Ala Asn Pro Gly Ser

485 490 495

Ser

<210> 2

<211> 3282

<212> DNA

<213>Arabidopsis arabidopsis（Arabidopsis thaliana）

<400> 2

gtagttgcaa acaccgatca gtcacagaca gtagcctctc accatcacca ccacggcggc 60

cgtcaccgac gccgatacac catgaaatta catttacacc ctctctcact tgtcttcttc 120

tttttcttct tcttcttccg accgacaatg tcgtcgaacc agcacgatta ctccgacgca 180

ctctcaaaat caattctctt cttcgaaggc cagcgctccg gttacctccc aaacgaccag 240

cgtatgacct ggcgccgtaa ctccggtctg agcgacggtt ggacacacaa catcgattta 300

accggcggtt attacgacgc cggagacaac gtcaaattca atttcccgat ggctttcacc 360

accacaatgc tcgcctggag cgtaattgag ttcggcgaat tcatgccttc gtcggagcta 420

cgtaactctc tcgtggctct ccggtggagc tctaattacc tcctcaaatc cgtttcacag 480

ctacctaacc ggatcttcgt ccaggttagt gtggttttaa tttgccaatt caatttcgta 540

actgtttttt tttatgccgg tgagtggcat ttttagtgaa tacgaaacta tggggaaggg 600

taaattgggg aattaaaata acacagagat tgaggaaact gaatcgtttg agttgccaag 660

agatgaaacg aacccgtgaa gtgggtgttt gtttttggac tctcaagagt ctctctgcat 720

ttaccttttt tgtcgttgcg tcacactttt tttggccctt tctattttcc tatatattct 780

tttctttcta cgaaattatc aatacacatc aatatcaatt taattagtta ttgttaatga 840

caaagtccag tacgtaacat acgtataaca tcgaatgaat catttaggaa ataacaattg 900

actaggaact ttataagttg gcaacgtaaa tgtcaattta attttggtct tattttgtgt 960

gtgtcccgta ctttgtagta aatacaaaga tgaccaacca ttagttttag ttgcagggtt 1020

ttttcgtttg tttttatgtt tattttttta aaaaaactac aacaataaat tttgttgact 1080

taaattcagc aataaatgga taatgataat tcaatagtac acctctaaaa taaattagtg 1140

aatgggttat gaaattatca acattgattc aatggtcgtt tgcatcataa ttaataaatt 1200

atgatcattt aataattatt agagttgaga tacgatgata gagacttctt tctttcagaa 1260

tcgttcaatt ctctttgttc caatattaga aaaatcaaac atcagatatg atgttgatgt 1320

tgttaggtgc atttcttatt ttattcattg ataatttcat atcttgattt atatattgtt 1380

ttatttagag ggcttgaata tttaatattt ttgaaaacta actaaggtag gaaaaaaata 1440

aacgaacttg tgataggtag gtgatccgat agcagatcat aattgctggg aaagaccaga 1500

agacatggat acaccacgga ctgcttacgc cgttaatgcc ccgaatccgg cttctgaagt 1560

tgccggagaa actacggcgg ccctctcagc tgcttcgatt gctttccgat catcggatcc 1620

aggatactct caaacattgc tccaaaatgc tgtcaaaact tttcagttcg ctgatatgta 1680

tcgcggtgct tatagcagca atgacgatat caaaaatgat gtgtgtcctt tctactgcga 1740

tttcaacgga tttcaggtaa acatcaatcc caacatttgt gtgactaaag aagttatcaa 1800

atatttaaca ggatcattag aacaagctaa atatctatac attcttggta aatattatat 1860

tttgtacatt ttcttggtga tgattttctt taatgttgtt ttttgtaaaa tttttgttat 1920

gggtaggatg agttattgtg gggagcagct tggctaagaa aagcgactgg tgatgaaagc 1980

taccttaatt acatagagag taaccgtgaa ccgtttggtg ctaacgataa cgtagacgaa 2040

tttggctggg acaacaaagt tggtggactt aatgttctcg tttcgaaggt tagttcaata 2100

cttccaattt tattaacatc aaatttaggg gatgaaatcg aaattaactt taaatacttt 2160

agcaaaagtt tagtaaacac gaggctttca cgtgcatatc aaagttatgt tcttttcttt 2220

ccgtgtacgt gaatccaaac tgacccggtt aagaaccaaa ccggtaccat aactgataaa 2280

cttggtggtt tgatttaatt tcggtacagg aagttattga aggaaatatg tacaacttag 2340

aggcgtacaa ggcatcagca gagagcttca tgtgtagtct ggtcccggaa tcttcgggac 2400

cgcatgttga gtatacttcc gctggtctac tttacaagcc cggcggtagc cagctccaac 2460

atgcgaccac catatctttc ctcctcctcg tatacgctca atatctttca agaagctctc 2520

tctccctcaa ttgcggcacc cttaccgtcc ctcctgatta tctccgccgt cttgccaaga 2580

aacaggttag accggtttga gttggtttgg tttaagttta taagtgaata acatataatt 2640

tgtttttggc atccagcaaa actaatcatg tatttattgg aaaatgaagg tggactatat 2700

attggggaat aatccgatgg gattatcgta catggtcgga tacggagaga gatatccaaa 2760

gaggatccat caccgtggct catcattgcc atcgattgtg gaccatccag aagccatcag 2820

atgcaaagac gggtcggttt atttcaattc taccgaaccg aacccgaacg ttttgattgg 2880

agcggtggtg ggtgggcccg gagaggacga tatgtacgat gatgatcggt ccgatttccg 2940

caagtccgag cccactactt acatcaatgc tcctttcgtt ggcgttttgg cgtactttgc 3000

agccaatcct ggttctagct aagctgacga catgggagta aaattggaaa acattattct 3060

taagttccaa gagtatggga gttctttttt ttcttacaag tctctcatct tcttgcccaa 3120

gtaaagaagg aaaaagttct tacagttaaa atagtttttc ttttacgcaa aattaatctg 3180

aataacttat tcggattatt cacctgttga tcatttgata cttctttttt tatcaaatgt 3240

aattaatagt ctattcatat atacatatat ataatggttg ct 3282

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223>IND identifies sense primer

<400> 3

cacccataca atcttggatc g 21

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223>IND identifies anti-sense primer

<400> 4

tgttctggtt gggttcaaaa g 21

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223>SALK_058083 inserts primer

<400> 5

tggttcacgt agtgggccat cg 22

<210> 6

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<223>CEL6 cloned upstream primers

<400> 6

ggactcttga ccatggtagt tgcaaacacc gatc 34

<210> 7

<211> 44

<212> DNA

<213> Artificial Sequence

<220>

<223>CEL6 cloned downstream primers

<400> 7

atttaccctc agatctacca tagcaaccat tatatatatg tata 44

<210> 8

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223>M13 sense primers

<400> 8

gagcggataa caatttcaca cagg 24

<210> 9

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223>M13 anti-sense primers

<400> 9

cgccagggtt ttcccagtca cgac 24

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223>CEL6 quantitative PCR sense primers

<400> 10

tttcccgatg gctttcacca 20

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223>CEL6 quantitative PCR anti-sense primers

<400> 11

gagagccacg agagagttac g 21

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223>Internal reference actin8 sense primers

<400> 12

taaggtcgtg gcaccacccg 20

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223>Internal reference actin8 anti-sense primers

<400> 13

atccgagttt gaagagctac aa 22

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223>CEL6 identifies sense primer

<400> 14

cgtattatcc aagcataaat cgc 23

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223>CEL6 identifies anti-sense primer

<400> 15

tagctgtgaa acggatttga gg 22

<210> 16

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223>SALK_060505 Insert Fragment primers

<400> 16

tggttcacgt agtgggccat cg 22

<210> 17

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<223>WiscDsLox485-488K15 Insert Fragment primers

<400> 17

aacgtccgca atgtgttatt aagttgtc 28

Claims (10)

1. a kind of cellulase, it is characterised in that its amino acid residue sequence is such as shown in (a) or (b)：

(a) such as SEQ ID No：Amino acid residue sequence shown in 1；

(b) to such as SEQ ID No：Amino acid residue sequence shown in 1 by 1 or several amino acid residues substitution and/or Missing and/or addition and the amino acid sequence with regulating and controlling effect that ftractureed to plant silique.

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

3. the encoding gene of cellulase according to claim 2, it is characterised in that its nucleotide sequence such as (a), (b), (c) or shown in (d)：

(a) such as SEQ ID No：Polynucleotides shown in 2；

(b) coding such as SEQ ID No：The polynucleotides of amino acid shown in 1；

(c) with SEQ ID No：Polynucleotides shown in 2, which have more than 80% homology and had to plant silique cracking, to be adjusted The nucleotide sequence of control effect；

(d) under high high stringency conditions with SEQ ID No：The nucleotide sequence of the 2 DNA sequence dna hybridization limited.

4. the recombinant vector containing encoding gene described in Claims 2 or 3.

5. the recombinant microorganism containing encoding gene described in Claims 2 or 3.

6. the transgenic cell line containing encoding gene described in Claims 2 or 3.

7. the expression cassette containing encoding gene described in Claims 2 or 3.

8. application of the cellulase in regulation and control plant silique cracking gene described in claim 1.

9. application of the cellulase according to claim 8 in regulation and control plant silique cracking gene, it is characterised in that：Institute Stating plant includes monocotyledon and dicotyledon.

10. application of the cellulase according to claim 8 in regulation and control plant silique cracking gene, it is characterised in that： The plant is arabidopsis or rape variety.