CN104480121B - Control ZmCLA1 genes and its method in the seed selection type of resistance to more seedlings corn and the application of maize leaves corner dimension - Google Patents
Control ZmCLA1 genes and its method in the seed selection type of resistance to more seedlings corn and the application of maize leaves corner dimension Download PDFInfo
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Abstract
The invention discloses the ZmCLA1 genes of control maize leaves corner dimension, its nucleotide sequence such as SEQ ID NO：Shown in 1.The present invention uses forward genetics method（By build mapping population Leaf angle is carried out QTL be tentatively set to, finely positioning）Candidate gene is accurately separated, the function of ZmCLA1 genes does not have any report in plant, the invention provides the gene ZmCLA1 and its encoding proteins of regulation and control maize leaves corner dimension.The gene has important value for the molecule mechanism scheduling theory research that parsing corn Leaf angle is formed, and will contain SEQ ID NO：The gene of sequence shown in 1 is transformed, and is imported in milpa, can create the corn breeding material that plant type is compact, Leaf angle is small, has important practical application meaning to corn breeding.
Description
Technical field
The present invention relates to corn breeding technique field, and in particular to control maize leaves corner dimension ZmCLA1 genes and its
Method and application in the seed selection type of resistance to more seedlings corn.
Background technology
Corn is the second largest cereal crops of China, 25,450,000 hectares or so of long-term cultivated area, sown area and total yield
Account for 25% and 28% of cereal crops or so.As the reduction of cultivated area, population are continuously increased, corn consumption figure is then in firm
Property increase.Therefore, improve per unit area yield, increase the important goal that total yield is China's corn breeding long-sought.Study both at home and abroad and show,
It is the major measure for developing Maize Production to improve per unit area yield, and the raising of per unit area yield depends on the seed selection of new varieties and updates.With
Improving the improvement of the corn variety for the purpose of yield is realized by improving hybrid vigour and plant type improvement.
20 beginnings of the century were always the world to be improved as the maize genetic of target using hybrid vigour seed selection new varieties so far
The important directions of various countries' research of agricultural science, but with the raising being bred as with commercial variety yield level, it is simple excellent by hybrid
Snobbish technology has been difficult to realize the target of corn Sustainable high yield.Practice at home and abroad proves, continuous with yield levels of maize
Lifting is popularized with Maize Production mechanization, and raising corn planting density has turned into realizes that corn Sustainable high yield is most important
Technical measures.In the past few decades, the corn hybrid seed single plant yield of upper application produced in USA is not significantly improved, and single
Produce and the main cause of total yield raising is the raising of planting density and the enhancing of resistance.U.S.'s average yield per mu in 2009 is approached
700 kilograms, planting density is at 5500 plants/acre or so.Compared with the U.S., the corn planting density and yield of current China are still
There is the space of further raising.For example, the producing region average yield per mu of China's Huang-Huai-Hai corn is less than 400 kilograms, planting density is
3500-4500 plants/acre.Therefore, it is to improve planting density to improve per unit area yield, the maximum potential of increase total yield.In maize population
Under high density adverse environmental factor, how milpa could fully receive and utilize lightHow milpa is to being produced under dense planting environment
Raw corresponding regulation and control react to improve photosynthetic efficiencyCorn shade syndrome（Shade Avoidance Responses, SAR）
High density adverse circumstance it occur frequently that a kind of phenomenon, when corn is under this adverse environmental factor, it will change original growth mould
Formula and Photosynthates translocation mode, ultimately result in yield reduction.The essence of corn High planting is the corn group under high concentration environment
Body photosynthetic efficiency is high, and shade syndrome is low, and colony is yield per unit area high.Therefore, the raising of corn planting population density is needed
There is the seed selection and application of the excellent high-yield variety of the type of resistance to more seedlings and Nai Mi characteristics.Compact and reasonable plant type is the one of density-resisting and high-yield
Individual important external morphology index, such as leaf angle, length, width, spatial distribution and attitude all have impact on the kind of maize population
Density in planting.Corn photosynthate source, stream, the reasonable high-efficiency operation in storehouse etc. are an important inherent physiological mechanisms of density-resisting and high-yield, control
The parsing of the clone and molecule mechanism of density-resisting and high-yield character gene processed, then be the basis for carrying out density-resisting and high-yield corn variety seed selection.
Therefore, parsed by the map based cloning to characters with plant gene and molecule mechanism, explore density-resisting and high-yield molecular mark
Technology, for the excellent high-yield corn kind of the seed selection type of resistance to more seedlings and Nai Mi characteristics provides theory and technology support.
Leaf angle is one of most important economical character of Plant Type in Maize, although being positioned about the QTL of maize leaves angle, is arrived
Also obtain corresponding candidate gene not over map-based cloning so far.And some genes related to corn Leaf angle
It is studied by comparative genomics and mutation body technique.Ku etc.（2011）Using comparative genomics method, clone and be located at
The candidate gene of the qLA2 on the second chromosome.Research shows, compact parental inbred line Henan 82 and loose type parental inbred line
Its 5 '-UTR end of Shen 137 " CTCC " is changed into " CCCC ", have impact onZmTAC1Expression so that further influence Leaf angle
Size.It is indicated aboveZmTAC1The height of gene expression dose is the change by 5 '-UTR site sequences ' CTCC '-' CCCC '
Change regulation and control.Moreno etc.（1997）It is rightlglThe research of mutant finds that the mutant isligulelesslGene expression lacks
Lose mutant, the mutant shows as being formed the tip of a leaf and auricle, the junction of blade and leaf sheath can not develop.Using work
Agent (Ac) transposable element, will used as a molecular labellglAllelelgl-mlTherefrom separate and clone and, research
ConfirmLG1Gene produces function with a kind of cell-autonomous fashion.Juarez etc.（2004）It was found thatrld1Withlbl1Mutant is showed
Go out paraxial/upward leaf portion form.By cloning its corresponding gene discovery,rld1One HD-ZIPIII albumen of coding, leads to
The transcription cracking of the miR166-directed of shaft end too far defines space proximal ends expression.SemidominantRldl-OMutant exists
One, site of miR166 complementations mononucleotide is replaced and causes the continuous expression of the mutant transcript at distal shaft end, and this causes leaf inclined
To proximal ends.Genetic analysis showslbl1WithRldl-OSuppress each other, illustrate that this 2 genes are acted as in same path
With.It is rightyabbyThe research of gene finds that it directly causes lateral organ to outgrowth.Compared with the QTL for having identified, identify
Relevant with Leaf angle number gene it is far from enough, illustrate that the substantial amounts of candidate gene related to Leaf angle needs further mirror
It is fixed.The gene ZmCLA1 that this research is related to is that relevant with Leaf angle size found by map-based cloning is contained
The domain new gene of GHMP-kinases-N and Pmev-kin-ERG8, its Molecular biological function at present in plant still
Do not report.The present invention have studied the Molecular biological function of the gene regulation corn Leaf angle, and to cultivate the type of resistance to more seedlings and resistance to
The excellent high-yield corn kind of close characteristic provides a potential new gene resource.
The content of the invention
It is an object of the invention to provide a kind of gene ZmCLA1 of regulation and control maize leaves corner dimension, the gene has such as
SEQ ID NO：DNA fragmentation shown in 1.The present invention also includes such as SEQ ID NO：Protein sequence shown in 2.The gene
The reduction of mRNA accumulations, causes corn Leaf angle to reduce, therefore the clone of the gene helps to understand what corn Leaf angle was formed
Molecular mechanism.
The present invention utilizes RNAi（RNA is disturbed）Technology, suppresses the expression of the endogenous ZmCLA1 genes of corn, causes cross section thin
The length of born of the same parents shortens, and causes Leaf angle to reduce（See embodiment 6）.It is exactly specifically by ZmCLA1 genes and other regulation and control units
Part, such as constitutive promoter（Such as CaMV35S promoters）Or organ specific promoters construct gene and suppress expression vector,
By transgenic technology（Such as antisense RNA or RNAi）The corn inbred line that Leaf angle is small, plant type is compact is created, for cultivating corn
Resistance to close new varieties.
The technical scheme is that：Control the ZmCLA1 genes of maize leaves corner dimension, its nucleotide sequence such as SEQ
ID NO：Shown in 1.
Control the ZmCLA1 genes of maize leaves corner dimension, its protein sequence such as SEQ ID NO：Shown in 2.
Control maize leaves corner dimension ZmCLA1 genes the seed selection type of resistance to more seedlings corn method, it the step of it is as follows：
（1）With compact self-mating system Henan 82 as donor parents, with loose type corn inbred line Shen 137 as receptor parent, adopt
The NIL Shen 137- of the main effect qLA1 positioned at the first chromosome is built with backcross transformation binding molecule marker assisted selection
NIL68；
（2）ZmCLA1 genes are separated using map-based cloning, the endogenous ZmCLA1 genes of corn are suppressed using RNAi technology
The expression small self-mating system of selecting and breeding corn Leaf angle.
The ZmCLA1 genes of maize leaves corner dimension are controlled to be applied to the seed selection type of resistance to more seedlings corn, in the volume of ZmCLA1 genes
Design a pair of RNAi primers in code region
RNAi-F：5’-actagtggatccgttgttgcagttcttcttc-3’
RNAi-R：5’-agatctggtaccggatagcactaccgactcat-3’
From pBI1391-ZmCLA1In amplify, forming hairpin structure with the sequence and its reverse sequence is inserted into
In pJL1460 carriers, so that pZmCLA1-RNAi carriers are formed, by recombinant plasmid transformed Agrobacterium competent cell, for turning
Change corn inbred line Shen 137, obtain the transgenic positive strain that Leaf angle shows different degrees of reduction.
Specific method of the present invention is as follows：
1. the present invention, with compact self-mating system Henan 82 as donor parents, is acceptor parent with loose type corn inbred line Shen 137
This, the NIL of the main effect qLA1 positioned at the first chromosome is built using backcross transformation binding molecule marker assisted selection,
We are named as Shen 137-NIL68, and the construction method of NIL is shown in detailed description in embodiment 1；
2. ZmCLA1 genes are separated using map-based cloning（See embodiment 2）.Using qRT-PCR technical Analysis
The expression rule of ZmCLA1, as a result shows that the expression quantity of the gene different times different parts in Shen 137 is significantly higher than Shen
Expression quantity in 137-NIL68, and a large amount expression in stem apex, blade, leaf sheath, pulvinus, illustrate ZmCLA1 positive regulation corns
Leaf angle（See embodiment 3）；
3. the present invention analyzes the cellular morphology in the maize leaf cross section of Shen 137 and Shen 137-NIL68 different times, knot
It is caused by the cell length of Transverse section of leaf blade reduces that fruit shows that the Leaf angle of Shen 137-NIL68 diminishes（See embodiment 4）；
4. overexpression Transgenic studies（See embodiment 5）Prove that the candidate gene ZmCLA1's that the present invention is obtained crosses scale
Up to the reason for causing corn Leaf angle to increase；
5. using the self-mating system that the expression selecting and breeding corn Leaf angle of the RNAi technology suppression endogenous ZmCLA1 genes of corn is small
（See embodiment 6）.
The beneficial effects of the invention are as follows：
1. the present invention uses forward genetics method（By build mapping population Leaf angle is carried out QTL be tentatively set to,
Finely positioning）Accurately separate candidate gene；
2. the function of ZmCLA1 genes does not have any report in plant, big the invention provides regulation and control corn Leaf angle
Small gene ZmCLA1 and its encoding proteins.The gene studies tool for the molecule mechanism scheduling theory that parsing corn Leaf angle is formed
There is important value；
3. SEQ ID NO will be contained：The gene of sequence shown in 1 is transformed, and is imported in milpa, can create strain
The corn breeding material that type is compact, Leaf angle is small, has important practical application meaning to corn breeding.
Brief description of the drawings
Sequence table SEQ ID NO:1 is the nucleotide sequence of the ZmCLA1 genes that the present invention separates clone, and sequence length is
2097bp, wherein 339-1878 are code area.
Sequence table SEQ ID NO:2 is the amino acid sequence of ZmCLA1 gene codes.
Fig. 1 Henan 82, Shen 137-NIL68 and the field phenotype in Shen 137, A in figure：Stationary stage（Corn Post flowering 10 days）
Henan 82（It is left）, Shen 137-NIL68（In）With Shen 137（It is right）Plant forms；B：Second, fringe top leaf Henan 82（It is left）, Shen 137-
NIL68（In）With Shen 137（It is right）Leaf angle；C：10 days leaf Henan 82 after pollination（It is left）, Shen 137-NIL68（In）With Shen 137
（It is right）Fringe top Leaf angle average value；
The Molecular Identification result of Fig. 2 map based cloning ZmCLA1 genes, a in figure：Using 3684 individual plants of BC3F2 colonies,
Screen 16 exchange individual plants, 16 are exchanged individual plants carry out label L AI25 that qLA1 QTL reduce by finely positioning with
In bnlg1484 is interval；b：Using BC4F2 colonies totally 8752 individual plants, 17 plants of exchange individual plants are screened altogether, exchange single to 17
Strain is carried out in label L AII40 and the LAII53 interval that finely positioning reduces qLA1 QTL；c：By finely positioning by qLA1's
Interval is narrowed down in the region of 30.26Kb, and the region includes 3 candidate genes.Black lines and red boxes represent base respectively
The introne and extron of cause；d：3 candidate gene GRMZM2G019260（ZmCLA1, it is left）、GRMZM2G019567（In）With
GRMZM5G808271（It is right）Expression quantity in Henan 82, Shen 137-NIL68 and Shen 137（MRNA accumulations）；
Fig. 3 is the expression pattern of ZmCLA1 genes, and qRT-PCR detects ZmCLA1 genes from the 3-19 leaf phases in Shen 137 and Shen
Expression quantity of the 137-NIL68 in blade（MRNA accumulations）, 18S genes are used as internal reference；
Fig. 4 is the expression pattern of ZmCLA1 genes, and qRT-PCR detects ZmCLA1 genes from the 3-19 leaf phases in Shen 137 and Shen
Expression quantity of the 137-NIL68 in leaf sheath（MRNA accumulations）, 18S genes are used as internal reference；
Fig. 5 is the expression pattern of ZmCLA1 genes, and qRT-PCR detects ZmCLA1 genes from the 3-19 leaf phases in Shen 137 and Shen
Expression quantity of the 137-NIL68 in pulvinus（MRNA accumulations）, 18S genes are used as internal reference；
Fig. 6 is the expression pattern of ZmCLA1 genes, and qRT-PCR detects ZmCLA1 genes from the 3-19 leaf phases in Shen 137 and Shen
Expression quantity of the 137-NIL68 in stem apex（MRNA accumulations）, 18S genes are used as internal reference；
Fig. 7 isZmCLA4The morphological observation of gene.In figure,（a）The pulvinus of Shen 137,（b）D132 pulvinus,（c）In figure（a）
The crosscutting covering weave at position pointed by middle arrow,（d）In figure（b）The crosscutting covering weave at position pointed by middle arrow；
Fig. 8 is the plant phenotype that mCLA1 overexpression transgenosis makes the Leaf angle increase of self-mating system Henan 82, A in figure：Stationary stage
Overexpression ZmCLA1 positive plants（It is left）And adjoining tree（It is right）Form；B：Stationary stage overexpression ZmCLA1 positive plant
（It is left）And adjoining tree（It is right）Fringe top Leaf angle average value；
Fig. 9 is that the expression for suppressing endogenous ZmCLA1 in self-mating system Shen 137 using RNAi technology can make what Leaf angle reduced
As a result, A in figure：Stationary stage RNAi-ZmCLA1 positive plant（It is left）And adjoining tree（It is right）Form；B：Stationary stage
RNAi-ZmCLA1 positive plants（It is left）And adjoining tree（It is right）Fringe top Leaf angle average value.
Specific embodiment
The present invention is using the compact corn self-mating system Henan 82 of Agricultural University Of He'nan's seed selection（Fig. 1）It is female parent, with Shenyang
The loose type corn inbred line Shen 137 of Academy of Agricultural Sciences's seed selection（Figure）For paternal hybrid obtains F1, F1 selfings obtain F2 mapping groups
Body and its derivative F2:3 familys are target group, by 3 Leaf angle phenotypic evaluations in place, QTL have been carried out to Leaf angle
Positioning, common location is to 3 QTLs relevant with Leaf angle, wherein the effect value of the main effect qLA1 on the first chromosome is maximum
（Ku et al. 2010）.Therefore, with self-mating system Henan 82 as donor parents, self-mating system Shen 137 is that receptor parent builds qLA1's
NIL.Built-up using backcross transformation binding molecule mark auxiliary selection method, the NIL of structure is Shen
137-NIL68（Fig. 1）, to further understand present disclosure and purpose, the following examples 1 will be described in detail of the invention
Particular technique implementation steps.
1：The structure of NIL
With Leaf angle is small, plant type is compact corn inbred line Henan 82 as donor parents, the corn that Leaf angle is big, plant type is loose
Self-mating system Shen 137 is that receptor parent passes through backcross transformation binding molecule marker assisted selection technique construction NIL（Fig. 1）.
Spring in 2007 assembles acquisition F in the parent of Zhengzhou two1；Winter in 2007 plants F in Hainan1Generation and receptor parent Shen 137, with Shen 137 times
Hand over F1Obtain BC1F1；According to BC1F1Phenotypic Selection Leaf angle small 13 plants in the spring in 2008 in Zhengzhou kind into head progeny row, continue with
The backcrossing of Shen 137 obtains BC2F1；Therefrom 15 small individual plants of selection Leaf angle continue in the winter in 2008 in Sanya, Hainan kind into head progeny row
BC is obtained with the backcrossing of Shen 1373F1；Spring in 2009, by BC3F1In Zhengzhou, kind is into head progeny row, from BC3F1It is middle big according to plant Leaf angle
Bilateral mark small, with reference to qLA1 carries out genotyping, obtains 14 exchange individual plants（And 14 are exchanged with individual plant using structure
222 SSR markers of F2 genetic maps carry out Analysis of Genetic Background.Wherein bilateral mark and 222 SSR markers and reference storehouse are beautiful
Rosy clouds, Plant Type in Maize correlated traits molecular genetic study mechanism, Agricultural University Of He'nan Ph.D. Dissertation, 2010）Continue and Shen 137
Backcrossing obtains BC4F1, while obtaining BC3F2 to exchanging individual plant selfing；Winter in 2009 in Sanya, Hainan, by BC4F1 and BC3F2 kinds
Plant, according to BC4F1The phenotype combination genotype of colony have selected 22 plants and exchange individual plant selfing acquisition BC4F2；2010 spring will in Zhengzhou
Obtain BC4F2 kinds to plant, according to BC4F2The phenotype combination genotyping of colony, obtains 11 exchange individual plants, and 11 are exchanged
Individual plant carries out Analysis of Genetic Background using above-mentioned 222 SSR markers, and therefrom select genetic background response rate high 3 exchange single
Strain selfing, and then obtain in the site homozygosis, the NIL of inheritance stability.
2：ZmCLA1 genes are separated using map-based cloning
Using BC3F23684 plants of colony therefrom selects individual plant of the Leaf angle less than or equal to 12 ° as finely positioning colony
There is strain 668, useqLA1The mark bnlg1803 and bnlg1484 of bilateral analyze its genotype, therefrom select in bnlg1803 or
There is restructuring at bnlg1484 and exchange 6 plants of individual plant；Individual plant 812 plant of the simultaneous selection Leaf angle more than more than 20 °, and binding molecule
Mark selection filters out 10 plants of big exchange individual plants of Leaf angle.Using it is newly developed and with 9 couples of strong primer LAI5 of polymorphism,
LAI8、LAI9、LAI10、LAI18、LAI25、LAI29、LAI31、LAI38（Marker development method is with reference to Mr. Zhang, corn Leaf angle
Relevant ZmCLA4 genes map based cloning and functional analysis, Agricultural University Of He'nan Ph.D. Dissertation, 2014）Analysis exchanges individual plant
Genotype.According to exchange individual plant target zone marker genetype, can qLA1 be limited to label L AI25 and bnlg1484 it
Between (Fig. 2 a).
In order to further determine that qLA1 for one or more candidate genes, by the use of 8752 plants of BC3F2 colonies as finely positioning
Colony, therefrom selects Leaf angle to have 1682 plants less than or equal to 12 ° of individual plants, with the label L AI25 of qLA4-1 bilaterals with
Bnlg1484 analyzes its genotype, therefrom selects restructuring 11 plants of individual plant of exchange at SR4-6 or SSR4-39；Simultaneous selection
Simultaneously the selection of binding molecule mark filters out 6 plants of big exchange individual plants of Leaf angle to 1769 plants of individual plant of the Leaf angle more than more than 20 °.Again
Using with strong 9 couple of polymorphism mark newly developed（LAII10、LAII26、LAII31、LAII33、LAII36、LAII39、
LAII40、LAII42、LAII53）Analysis exchanges individual plant genotype.The small Leaf angle for the exchanging individual plant variation model of 11 plants of Leaf angles
It is 6.70 ° -12.30 ° to enclose, and average Leaf angle is 10.67 °, and the Leaf angle range of variation of the big exchange individual plant of 5 plants of Leaf angles is
20.12 ° -21.35 °, average Leaf angle is 20.85 °.According to the marker genetype for exchanging individual plant target zone, can be qLA1
It is limited between label L AII40 and LAII53（Fig. 2 b）.The interval about 30.26-kb, comprising three predicted genes (Fig. 2 c),
Therefore using the 30.26-kb regions between label L AII40 and LAII53 as the gene region of qLA1.With Henan 82, Shen
137th, the mRNA reverse transcriptions of Shen 137-NIL are template into cDNA, 3 candidate genes are separated, while with the DNA of above-mentioned 3 materials
It is template, separates 3 initiating sequences of candidate gene.Shown by sequence difference analysis result, GRMZM2G019260 starts
Subsequence has differences in Henan 82, Shen 137 and Shen 137-NIL68, and other 2 gene all sequences all do not exist difference.Say
It is difference due to GRMZM2G019260 in promoter sequence that the Leaf angle size of bright Shen 137 and Shen 137-NIL68 has differences
Cause.Further to verify this conclusion, using qTR-PCR technologies（With reference to Mr. Zhang, the relevant ZmCLA4 genes of corn Leaf angle
Map based cloning and functional analysis, Agricultural University Of He'nan Ph.D. Dissertation, 2014）With the Henan 82 of 5 leaf phases, Shen 137, Shen 137-
NILs is stem apex material, extracts mRNA and reverse transcription into cDNA；Take these cDNA as the table of above-mentioned 3 candidate genes of template analysis
Up to amount, as a result show, GRMZM2G019260 has significant difference in 3 materials, though GRMZM2G019567 is in 3 materials
So express but in the absence of significant difference, GRMZM5G808271 is not expressed in 3 materials（Fig. 2 d）.These results show the He of Shen 137
Shen 137-NIL68 Leaf angles have differences to be caused by the expression quantity difference of GRMZM2G019260, is thus illustrated
GRMZM2G019260 is exactly the candidate gene for regulating and controlling corn Leaf angle in qLA1 regions, is named as ZmCLA1.
3：ZmCLA1 positive regulation corn Leaf angles
ZmCLA1 increases Leaf angle in Shen 137, and Leaf angle is reduced in the 137-NIL68 of Shen, therefore we detect
The expression of ZmCLA1 genes.QRT-PCR testing results show, from the blade of 3 to 19 leaf phases of corn growth（Figure
3）, leaf sheath（Fig. 4）, pulvinus（Fig. 5）And stem apex（Fig. 6）In high efficient expression.In the different developmental phases of corn, Shen 137 and Shen
The expression quantity of 137-NIL68 has certain similitude, is expressed since 3 leaf phases, and 9 to 10 leaf phase expression quantity are higher, and 11
Expression quantity reduction after the leaf phase.For different materials, ZmCLA1 is obvious in the expression quantity of the different times in Shen 137, different parts
Higher than Shen 137-NIL68（Fig. 3-6）, show the Leaf angle just relative increase of the expression quantity corn high of ZmCLA1, thus propose
ZmCLA1 as positive regulation cytokine regulatory corn Leaf angle size.
4：Cellular morphology observation proves that the Leaf angle of Shen 137-NIL68 is due to the contracting of the position such as pulvinus cross section cell length
Caused by small, embodiment 3 pairsZmCLA1Gene Different Organs, expression pattern of different leaf phases in Shen 137 and Shen 137-NIL68 enter
Row analysis, as a result shows,ZmCLA4Gene in stem apex, pulvinus, leaf sheath, blade equal high efficient expression therefore, using paraffin section
Method（With reference to Pan Ye etc., a kind of quick paraffin method suitable for plant tissue.2008, agriculture as the foundation of the economy science, 24（3）112-
Method shown in 115 and step）The eucaryotic cell structure analysis of pulvinus, blade position in stationary stage, helps to understandZmCLA1Base
Because of the analysis in expression effect herein.By to pulvinus, Leaf cell structure it has been observed that Shen 137 and Shen 137-NIL68 plant
There is significant difference in the length of the cross section cell at its selected position in strain.As seen from Figure 7, Shen 137-NIL68
Cell be significantly shorter than Shen 137.These results show that ZmCLA1 may influence cell development so as to cause the change of Leaf angle.
5：ZmCLA1 overexpression transgenosis verifies its gene function
（1）The structure of over-express vector
According toZmCLA1Coding region sequence design the pair of primers TF and TR of gene, the end of primer 5 ' respectively plus BglII andSpeI restriction enzyme sites.With in the primer amplification material of Shen 137ZmCLA1CDNA sequence, PCR primer agarose gel electrophoresis inspection
Survey, recovery purpose PCR primer is connected into pMD18-T carriers, convert, select monoclonal, PCR detections, sequencing and will be sequenced correctly
Bacterium solution extract plasmid, by the plasmid and pBI1391 vector plasmids respectively with BglII andSpeI restriction enzyme sites carry out double digestion.
The purpose fragment after digestion is separately recovered, carrier segments and purpose fragment, connection product conversion large intestine are connected using T4 ligases
Bacillus, identifies positive colony, extracts recombinant plasmid, is named as pBI1391-ZmCLA1.By the impression of recombinant plasmid transformed Agrobacterium
State cell, for maize transformation self-mating system Henan 82.
Primer TF：'5-GAAGATCTATGGAAGTGGTGGCGTCG -3', horizontal line part is restriction enzyme site BglII
TR：5'-GGACTATCAGTATTAATCTGAATGGAC -3', horizontal line part is restriction enzyme siteSpeI
（2）Genetic transformation
Using agriculture bacillus mediated corn stem apex genetic transforming method（With reference to Mr. Zhang, the relevant ZmCLA4 of corn Leaf angle
Gene map based cloning and functional analysis, Agricultural University Of He'nan Ph.D. Dissertation, 2014）.Sprouted by Seed Treatment, seed,
Cut growing point, bacterium solution culture, contaminate, the seedling contaminated, 23 degree of cultures can just be moved into basin for three days, and running water is used before transplanting seedlings
Seedling is washed, seedling is moved into flowerpot after cleaning.After transplanting, one layer of vermiculite for slightly moistening is spilt above, flower is covered on dipped preservative film
On basin（Per 60 plants of basin）, cardboard shading is covered, when waiting seedling long to two one cores of leaf, spray herbicide is uniform during sprinkling, greenhouse
Middle temperature is at 30 degree or so.The seedling to herbicide sensitive is rejected, the seedling to herbicide insensitiveness extracts blade STb gene, carries out
The PCR detections of marker gene, the positive strain to detecting transplants crop field, and selfing obtains T0 for seed, completes to transgenosis T0 generations
Qualification process.
The present embodiment obtains 23 plants of independent transgenic positives strains altogether, and its Leaf angle shows different degrees of increase, and 5 plants
Then Leaf angle is not changed significantly for the control strain of empty carrier pBI1391（Fig. 8）
6：RNAi（RNA is disturbed）Carrier conversion checking ZmCLA1 gene functions
According to the structure of ZmCLA1 genes, we devise a pair of RNAi primers in the coding region of ZmCLA1 genes
RNAi-F（5’-actagtggatccGTTGTTGCAGTTCTTCTTC-3’）And RNAi-R（5’-agatctggtaccGGATAGCACTACCGACTCAT-3 ', underscore therein represents the restriction enzyme site for adding）From pBI1391-ZmCLA1In amplify, forming hairpin structure with the sequence and its reverse sequence is inserted into pJL1460 carriers, so as to be formed
PZmCLA1-RNAi carriers.By recombinant plasmid transformed Agrobacterium competent cell, for maize transformation self-mating system Shen 137.It is lost
Pass genetic transformation of the method for transformation with embodiment 5.The present embodiment obtains 18 plants of independent transgenic positive strains, its Leaf angle table altogether
Now different degrees of reduction, and the control of 7 plants of empty carrier pJL1460 strain then Leaf angle is not changed significantly（Fig. 9）.
As can be seen here, by after the expression of endogenous ZmCLA1 genes in RNAi technology suppression Shen 137, causing corn Leaf angle
It is substantially reduced, it was demonstrated that thinking the expression of control ZmCLA1 genes can apply the self-mating system conduct for cultivating that plant type is compact, Leaf angle is small
The basic material of breeding, can be used to cultivate resistance to close breeding cross kind popularization and application in production.
Claims (2)
1. using the method for the ZmCLA1 gene seed selection type of the resistance to more seedlings corns for controlling maize leaves corner dimension, the control maize leaves
The nucleotide sequence of the ZmCLA1 genes of corner dimension such as SEQ ID NO：Shown in 1, the ZmCLA1 of maize leaves corner dimension is controlled
The protein sequence of gene such as SEQ ID NO：Shown in 2, it is characterised in that it the step of it is as follows：
（1）With compact self-mating system Henan 82 as donor parents, with loose type corn inbred line Shen 137 as receptor parent, using return
Transformation binding molecule marker assisted selection is handed over to build the NIL Shen 137- of the main effect qLA1 positioned at the first chromosome
NIL68；
（2）ZmCLA1 genes are separated using map-based cloning, the table of the endogenous ZmCLA1 genes of corn is suppressed using RNAi technology
Up to the small self-mating system of selecting and breeding corn Leaf angle.
2. application of the ZmCLA1 genes of control maize leaves corner dimension in the seed selection type of resistance to more seedlings corn, the control maize leaves
The nucleotide sequence of the ZmCLA1 genes of corner dimension such as SEQ ID NO：Shown in 1, the ZmCLA1 of maize leaves corner dimension is controlled
The protein sequence of gene such as SEQ ID NO：Shown in 2, it is characterised in that：Designed a pair in the coding region of ZmCLA1 genes
RNAi primers
RNAi-F：5’-actagtggat ccgttgttgc agttcttctt c-3’
RNAi-R：5’-agatctggta ccggatagca ctaccgactc at-3’
From pBI1391-ZmCLA1In amplify, forming hairpin structure with the sequence and its reverse sequence is inserted into pJL1460
In carrier, so as to form pZmCLA1-RNAi carriers, by recombinant plasmid transformed Agrobacterium competent cell, for maize transformation from
Friendship is Shen 137, obtains the transgenic positive strain that Leaf angle shows different degrees of reduction.
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| CN106591325B (en) * | 2017-02-09 | 2019-06-21 | 河南农业大学 | A kind of method and its application using corn ZmDPS10-2 gene breeding early blossoming corn |
| CN109022450B (en) * | 2018-08-15 | 2021-09-24 | 河南农业大学 | ZmCL 2-1 gene for regulating and controlling included angle of corn leaves and application thereof |
| CN111484995B (en) * | 2019-01-25 | 2021-10-19 | 中国科学技术大学 | Application of maize ZmSET 1gene |
| CN112048512B (en) * | 2020-09-21 | 2022-09-02 | 河南农业大学 | Forward regulation factor for regulating corn leaf included angle and application thereof |
| CN113481315B (en) * | 2021-07-06 | 2022-05-17 | 齐鲁师范学院 | Molecular marker related to dwarfing of maize plants |
| CN114736280B (en) * | 2022-05-24 | 2023-03-24 | 中国农业大学 | Application of ZmROA1 protein in regulation and control of plant tolerance |
| CN114736914B (en) * | 2022-06-15 | 2022-08-23 | 中国农业科学院作物科学研究所 | ZmTGA4 gene and application thereof in adjusting and controlling corn leaf angle and increasing density and yield |
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| CN102234642A (en) * | 2010-04-23 | 2011-11-09 | 河南农业大学 | Molecular marker of major dominant quantitative trait loci (QTL) for regulating and controlling maize leaf angle and method and application thereof |
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Non-Patent Citations (4)
| Title |
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| The ZmCLA4 gene in the qLA4-1 QTL controls leaf angle in maize (Zea mays L.);J. Zhang1,et al;《Journal of Experimental Botany》;20140701;第65卷(第17期);5063–5076 * |
| XM_008649643;genbank;《GENBANK》;20140802;1 * |
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