CN104650205A

CN104650205A - Protein for controlling rice leaf morphology and root hair development, and coding gene and application thereof

Info

Publication number: CN104650205A
Application number: CN201510071890.XA
Authority: CN
Inventors: 郭旻; 严长杰; 李传友
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2015-02-11
Filing date: 2015-02-11
Publication date: 2015-05-27

Abstract

The invention relates to a protein DNL1 for controlling rice leaf morphology and root hair development, and a coding gene and application thereof. The rice DNL1 is a protein as the following 1) or 2): 1) protein with the amino acid sequence disclosed as SEQ ID No.2; and 2) protein subjected to substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid residues sequence disclosed as SEQ ID No.2 in the sequence table with the same activity as SEQ ID No.2. The invention also discloses a coding gene of DNL1, and an expression box, a transgenic cell line, a recombinant bacterium and the like containing the gene. The gene has important theoretical values for leaf and root hair generation mechanisms, and can be used in rice breeding to enrich the genetic diversity of the existing rice varieties. The rice DNL1 protein can be used for cultivating the rice narrow leaf species and forming ideal rice plant types.

Description

The albumen and encoding gene thereof and application controlling rice leaf form and roots development

Technical field

The invention belongs to molecular biology of plants technical field, be specifically related to a kind of control rice leaf form and roots development albumen and encoding gene and application.

Background technology

Rice leaf carries out photosynthetic major organs, and its morphological specificity directly affects the plant type of paddy rice, and then affect the yield and quality of paddy rice.This research screens a dynamic narrow leaf mutant (dynamic narrow leaf 1, dnl1) in the mutant library taking indica rice 93-11 as background.Adopt map based cloning strategy, cloned DNL1 gene, obtained the encoding sequence of this gene in mutant.The clone of this gene and functional analysis are reported first in Indica Rice, understand the Regulation Mechanism of rice leaf and then be used to guide breeding and established important foundation for people.

Aux/IAA gene is growth hormone originally one of the responsive genes found the earliest, and the time that the albumen of its coding exists in vivo is very short, and the degraded of albumen is subject to the adjustment of growth hormone.Structural analysis shows, Aux/IAA albumen is made up of the structural domain of four high conservatives, and wherein structural domain II is relevant with the stability of albumen, structural domain I, III relevant with dimerization with IV.Aux/IAA albumen does not have the region with interaction of genes, therefore it forms heterodimer indirectly play transcripting regulating function by combining with auxin Response Factor (ARF): the structural domain I of ARF albumen can be transcribed in conjunction with adjustment with the promoter sequence of the growth hormone responsive genes in downstream, and when Aux/IAA albumen and ARF protein binding form dimer, ARF albumen just can not combine with the promoter sequence of downstream growth hormone responsive genes, thus have impact on transcribing of downstream growth hormone responsive genes.After being degraded under the induction of Aux/IAA albumen at growth hormone, ARF albumen just can be released.

In paddy rice, Aux/IAA gene family has 31 members (Jain et al., 2006), up to the present, only has OsIAA1, the getable research in detail of merit of several gene such as OsIAA11, OsIAA13, OsIAA23 and OsIAA31.The sudden change of these genes, all can affect the growth of Rice lateral root, but the mode of action played again different (Zhu et al.2012).Aux/IAA albumen function and regulatory mechanism in rice leaf is grown also is not found in the mutant found.

Summary of the invention

The object of this invention is to provide a kind of paddy rice DNL1 (dynamic narrow leaf 1, dynamic narrow leaf) albumen and encoding gene thereof and application.

Paddy rice DNL1 provided by the invention, derives from paddy rice, is following 1) or 2) protein:

1) protein be made up of the amino acid residue sequence of the SEQ ID № .2 in sequence table;

2) the SEQ ID № .2 amino acid residue sequence in sequence table had the protein of the identical activity of amino acid residue sequence of SEQ ID № .2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation.

In sequence table, sequence 2 is made up of 343 amino-acid residues.

For the ease of the purifying of DNL1, the aminoterminal of the protein that can form at the amino acid residue sequence by sequence 2 or carboxyl terminal connect label as shown in table 1.

The sequence of table 1 label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tagII	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned 2) DNL1 in can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.Above-mentioned 2) encoding gene of the DNL1 in is by the codon by lacking one or several amino-acid residue in the DNA sequence dna in sequence table shown in sequence 1 or 3, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence connecting the label shown in table 1 is held to obtain at its 5 ' end and/or 3 '.

The gene of above-mentioned paddy rice DNL1 of encoding also belongs to protection scope of the present invention.

Described paddy rice DNL1 albumen cDNA gene can be following 1) or 2) or 3) or 4) DNA molecular:

1) DNA sequence dna of SEQ ID № .1 in sequence table;

2) polynucleotide of SEQ ID № .2 protein sequence in polynucleotide;

3) DNA sequence dna limited with SEQ ID № .1 in sequence table has more than 90% homology, and coding identical function protein DNA sequence;

4) nucleotide sequence that the DNA sequence dna that can limit with sequence in sequence table 2 is under strict conditions hybridized.

Sequence 1 in sequence table is by 1032 based compositions, and its open reading frame (ORF) is from 5 ' end 1-1032 position Nucleotide.

Above-mentioned stringent condition can be at 0.1 × SSPE (or 0.1 × SSC), in the solution of 0.1%SDS, hybridizes and wash film under 65 DEG C of conditions.

Recombinant vectors containing above arbitrary described gene also belongs to protection scope of the present invention, as recombinant expression vector.

Available existing plant expression vector construction contains the recombinant expression vector of described gene.

Described plant expression vector comprises double base agrobacterium vector (as pBI121, pBin19, pCAMBIA2301, pCAMBIA3301, pCAMBIA1301-UbiN, pCAMBIA1300 etc.) and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor, as Agrobacterium crown-gall nodule induction (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean storage protein genes) 3 ' hold the non-translational region of transcribing all to have similar functions.

When using described gene constructed recombinant plant expression vector, any one enhancement type promotor, constitutive promoter or inducible promoter can be added before its transcription initiation Nucleotide, as the ubiquitin promoter (Ubiquitin), stress induced promoter Rd29A etc. of cauliflower mosaic virus (CAMV) 35S promoter, corn, they can be used alone or are combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.

For the ease of identifying transgenic plant cells or plant and screening, can process plant expression vector used, the coding can expressed in plant as added can produce enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, the antibiotic marker thing (gentamicin marker, kantlex marker etc.) with resistance or the chemical resistance reagent marker gene (as anti-weedkiller gene) etc. of colour-change.From the security consideration of transgenic plant, any selected marker can not be added, directly with adverse circumstance screening transformed plant.

Expression cassette containing above arbitrary described gene (DNL1), transgenic cell line and recombinant bacterium all belong to protection scope of the present invention.

The amplification total length of said gene or the primer pair of arbitrary fragment also belong within protection scope of the present invention.

Any one in described albumen, described gene, described recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium all can be applicable to cultivate narrow leaf and the less paddy rice of Gen Mao.

The carrier utilizing any one can guide foreign gene to express in plant, by the gene transfered plant cell of encoding said proteins, can obtain anti-salt, transgenic cell line that drought tolerance strengthens and transfer-gen plant.The plant tissue of conversion by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, conventional biology methods transformed plant cells or the tissue such as agriculture bacillus mediated, and is cultivated into plant by the expression vector carrying described gene.

The present invention obtains the narrow leaf mutant of indica rice 93-11, dnl1 (dynamic narrow leaf 1).This mutant not only significantly narrows at bearing desire performance blade, and does not have root hair, and lateral root number tails off.The present invention utilizes this mutant and japonica rice variety 9516 to hybridize and configures F ₂colony, adopts the method for map based cloning to be separated DNL1 gene, this genes encoding Aux/IAA6 albumen, controls the polarity growth of rice leaf and the morphogenesis of root.Up to the present, also do not control rice leaf form and raw the reporting for work of root hair about Aux/IAA6 simultaneously.After gene clone, not only to the genesis mechanism understanding blade and Gen Mao, there is important theory value, meanwhile, also can explore and be applied to rice breeding, enrich the genetic diversity of existing rice varieties.Paddy rice DNL1 albumen of the present invention can be used for cultivating paddy rice narrow leaf kind, moulds Ideal Rice Plant Type.

Accompanying drawing explanation

Fig. 1 different times leaf morphology compares.From the 9th leaf, blade significantly narrows, and recovers to the 14th leaf width of blade.

Fig. 2 mutant compares with wild-type (93-11) Root Morphology.Mutant is compared with wild-type, and lateral root number reduces, and does not have root hair.

Fig. 3 gene map based cloning.DNL1 gene is present in the interval of 20kb, only has an ORF predicted in this interval, encoding growth element response protein AUX/IAA6.

The Root Morphology of Fig. 4 process LAN and RNAi plant compares.9522 is contrast, OE-8 and OE-12 is process LAN plant; RNAi-15 and RNAi-16 is that RNA interferes strain.

Embodiment

Experimental technique in following embodiment, if no special instructions, is ordinary method.

Percentage composition in following embodiment, if no special instructions, is mass percentage.

Biological material source involved in the present invention is as follows:

Indica rice 93-11: Yangzhou research of agricultural science institute

Wuyunjing No.8, force educate No. 7, round-grained rice: Jiangsu (Wujin) rice research institute

Carrier pCAMBIA1301: open material, this carrier is purchased from Takara company

Agrobacterium EHA105: open material, this carrier is purchased from Takara company binary expression vector pTCK303: open material, this carrier is purchased from Takara company.

Embodiment 1: the phenotype of mutant dnl1 and genetic analysis

1. the phenotype analytical of mutant dnl1

Indica rice 93-11 obtains a seedling leaf by radiation and plant development bad but later stage is tending towards normal mutant strain, temporary called after dynamic narrow leaf mutant dnl1 (dynamic narrowleaf 1), the characteristic feature of dnl1 be blade at early growth period without considerable change, but significantly narrow mid-term in growth, anaphase blade recovers normal (Fig. 1).In addition, this mutant plant height becomes short, and each internode shortens all to some extent, and spike length shortens, and Secondary branch number tails off, and kernal number reduces, and setting percentage significantly reduces, and breeding time, delayed about 7-10 days, presented the hypogenetic feature of each side.In addition, root growth and the growth of dnl1 are obviously suppressed, and show as under water planting condition, 14 days later plant, side radical order and root approximate number amount, compared with wild-type, exist significantly and extremely significant difference, also can judge that this gene has pleiotropy (Fig. 2) thus.

2. the genetic analysis of mutant dnl1

With dnl1 and 93-11, and japonica rice variety Wuyunjing No.8, force educate the hybridization of No. 7, round-grained rice, its hybrid F ₁the leaf morphology of all acting normally, width of blade is similar to wild-type.At 3 F ₂in colony, all isolate normal leaf and dynamic narrow leaf plant two type, occur there are no intermediate type individuality.Show that dynamic narrow leaf proterties is recessive character relative to normal leaf.At F ₂(dnl1/93-11) in colony, the individuality of normal individual and mutant phenotype is respectively 127 and 35, meets the segregation ratio (χ of 3:1 ²=0.823< χ ² _0.05,1=3.84), show that this mutant character controls by 1 pair of recessive gene. but it is to be noted at other 2 F ₂in colony, isolated dynamic narrow leaf individuality is all fewer.In the F2 colony about having 1000 individual plants, only obtain 94 strains (dnl1 × Wuyunjing No.8) and 45 strains (dnl1 × force educates No. 7, round-grained rice) dynamically narrow leaf individuality respectively, seriously depart from Mendelian's single-gene segregation ratio (3:1), its reason may be relevant with the attribute of Xian Jing Jiao colony on the one hand, on the other hand also may be poorer than wild-type due to the vitality of mutant, in colony, be in competitive disadvantages, thus cause the individuality of mutation type surface significantly to reduce.

The acquisition of embodiment 2:DNL1 albumen and encoding gene DNL1 thereof

Utilize about 400 pairs of SSR marker to carry out polymorphism analysis to dnl1 and Wuyunjing No.8, it is 103 right wherein to disclose being marked with of two parent's polymorphisms, the F produced dnl1 and Wuyunjing No.8 hybridization with this 103 couple mark ₂in colony, the microcommunity of 10 strain exemplary dynamic narrow leaf individual plants compositions makes linkage analysis, found that microsatellite marker RM9, RM302 on the 1st karyomit(e) and dynamic narrow leaf the Characters chain.And then utilize and the 1st karyomit(e) shows between two parents polymorphic SSR and STS (Previous designs) mark to F ₂the dynamic narrow leaf plant of 94 strain of colony is analyzed.By the linkage group in the region at MAPMAKEER3.0 software building DNL1 gene place, DNL1 gene Primary Location marked between d15 and d20 at STS, the genetic distance marked with two is respectively 0.9cM and 2.2cM, with STS mark d17 show be divided into from.In order to further Fine Mapping DNL1, continue on the one hand to expand target group, dnl1 × force is educated the F that No. 7, round-grained rice is derivative ₂45 the recessive individual plants separated in colony mix with 94 recessive individual plants for gene Primary Location, and totally 139 recessive individual plants are used for the Fine Mapping of gene.Develop more STS at the section at target gene place on the other hand to mark.In this research, developed 49 STS mark, these marks are carried out polymorphism analysis to parent between RM9 and RM302, wherein 15 are marked between parent and show polymorphism (table 2).These 15 are utilized to mark two F further ₂dynamic narrow leaf individual plant in colony's (dnl1 × Wuyunjing No.8, dnl1 × force educate No. 7, round-grained rice) carries out marker gene type analysis, finds molecule marker M1-Z49, M1-Z41, M1-Z43 and d17 and ndl1 be divided into from, do not exchange individual plant appearance; And there are 1 and 2 exchange events respectively on M1-Z47 and M1-Z42 two mark seat, therefore, Dnl1 gene is limited between M1-Z47 and M1-Z42 by we.These two mark place PAC are respectively AP002972 and AP003768, and we construct the physical map covering Dnl1 gene accordingly, are successfully limited to by Dnl1 gene in the scope of about 172kb.

On this basis, dnl1/ Wuyunjing No.8 F is utilized ₃the normal individual plant of 500 strain in colony has carried out further Fine Mapping to DNL1, and development newly marks between M1-Z47 and M1-Z42.As a result, M1-Z40, M1-Z43 and DNL1 performance be divided into from, be thus positioned between M1-Z40 and M1-Z43 by DNL1, they are at a distance of about 20kb, construct with this physical map covering narrow leaf gene DNL1.(Fig. 3)

According to the gene prediction results of RGAP website, the location section of the 20kb at DNL1 place only exists the gene of a growth hormone response.This gene sequencing of this mutant and wild-type variety is analyzed, find that at three introns of dnl1, single base occurring replaces: T → C, T → A, A → T (lay respectively at AP003768:27596,27696,28018 places), exon there occurs single base and replaces A → G (being positioned at AP003768:28808 place).And there is a single base at promoter sequence to replace: the insertion of G → C and two base: AT.Sequencing result further demonstrate that the character of mutator gene, therefore this gene is tentatively decided to be the candidate gene of DNL1.Increased by cDNA, obtain the sequence 1 of DNL1 gene.Albumen prediction shows 343 amino-acid residues that sequence 1 is encoded as shown in sequence 2.

Table 2 is for the Molecular Marker Information of the assignment of genes gene mapping

Embodiment 3: the complementary assay of narrow leaf mutant dnl1 phenotype

1. the structure of complementing vector pDNL1

The restriction enzyme site analyzing the BAC clone covering DNL1 gene draws, utilizing Kpn I and Xba I double digestion BAC to clone (fine from Japan) can cut comprise the fragment of total length DNL1 gene, so design pair of primers, 5 ' end of front and back primer adds Kpn I and Xba I joint respectively, amplification DNL1 upstream region of gene (before initiator codon) 2.2kb, downstream (after terminator codon) 1.5kb, the fragment of overall length 5.9kb, is connected in the corresponding restriction enzyme site of carrier pCAMBIA1301.Select positive colony to extract plasmid DNA, form complementing vector pDNL1.

Complementary fragment primer:

DNLKpnF 5’-GGACTGGTACCTTCCAATCTGAAAGGAGGGAGGGG-3’(SEQ IDNO.33)

DNLXbaR 5’-CCTCATCTAGAGACCTTTCCTTTGTCCCCAGTT-3’(SEQ IDNO.34)

Front primer DNLKpnF 5 ' end adds Kpn I joint, and rear primer DNLXba I 5 ' end adds Xba I joint, and pcr amplified fragment size is 5.9kb.

2. the acquisition of transgenic line and phenotypic evaluation

Plasmid pDNL1 is imported Agrobacterium EHA105 by thermal shock, untransformed mutants dnl1 rataria.T ₀for the transfer-gen plant obtaining complemented phenotype, all similar wild-type 93-11 of all transfer-gen plant phenotypes, does not all grow narrow leaf in whole breeding time.In addition, the root phenotype of transfer-gen plant is also restored, and can observe root staple length and go out (Fig. 4).Illustrate that OsIAA6 is the candidate gene of DNL1.

The acquisition of embodiment 4, DNL1 gene overexpression and RNAi transfer-gen plant

The structure of DNL1 over-express vector: take 93-11cDNA as template, utilize combination of primers OE:F and OE:R, carry out pcr amplification, amplified production size is 1.5kb, carries out enzyme cut and be connected to the structure binary expression vector pTCK303 containing identical restriction enzyme site completing over-express vector DNL-OE with Sal I and Cla I.Plasmid DNL-OE is imported Agrobacterium EHA105 by thermal shock, transforms japonica rice wild-type variety force fortune round-grained rice No. 7 ratarias.T ₀for the transfer-gen plant obtaining roots development exception.The similar mutation type surface of root phenotype of two process LAN transgenic lines, growing of root hair receives suppression.

Process LAN fragment primer:

OE:F GCgtcgacCTCTTTCGTTCTCGCTTAG(SEQ ID NO.35)

OE:R CCatcgatTAGGCAAAATAGGGGTAGT(SEQ ID NO.36)

Front primer OE:F 5 ' end adds Sal I joint, and rear primer OE:R 5 ' end adds Cla I joint, and pcr amplified fragment size is 1.5kb.

Equally, we utilize PCR method, take cDNA as the DNA fragmentation of a template amplification 184bp, in two-way importing 1460 carrier, express with the Ubiquitin promotor promotor gene of corn.This plasmid vector is imported Agrobacterium EHA105, transforms japonica rice wild-type variety force fortune round-grained rice No. 7 ratarias.T ₀for the transfer-gen plant obtaining roots development exception.Lateral root number and the Gen Mao of two transgenic lines all there occurs change.

DNL1 process LAN and RNA interfere the phenotypic results of strain to show, the expression of rise or down-regulated gene all can affect the initiation and development of side root and root hair.

Claims

1. the dynamic narrow leaf protein D NL1 of paddy rice, is characterized in that, is following 1) or 2) protein:

2. a gene of the dynamic narrow leaf protein D NL1 of paddy rice described in claim 1 of encoding, is characterized in that, is following 1) or 2) or 3) or 4) DNA molecular:

1) DNA sequence dna of SEQ ID № .1 in sequence table;

2) polynucleotide of SEQ ID № .2 protein sequence in polynucleotide;

3. the recombinant vectors containing gene described in claim 2.

4. recombinant vectors as claimed in claim 3, it is characterized in that, be the recombinant expression vector containing gene described in claim 2 with existing plant expression vector construction.

5. recombinant vectors as claimed in claim 4, is characterized in that, described plant expression vector comprises double base agrobacterium vector and can be used for the carrier of plant micropellet bombardment.

6. the expression cassette containing gene described in claim 2.

7. the transgenic cell line containing gene described in claim 2 and recombinant bacterium.

8. gene described in claim 2 is cultivating the application in narrow leaf and the less paddy rice of Gen Mao.

9. the application of gene described in claim 2 in the transgenic cell line and transfer-gen plant of the anti-salt of cultivation, drought tolerance enhancing.

10. applying as claimed in claim 9, it is characterized in that, by carrying expression vector transformed plant cells or the tissue of gene described in claim 2, and the plant tissue of conversion being cultivated into plant.