CN107988234A - Application of the GRMZM2G701063 genes in environment stress - Google Patents

Application of the GRMZM2G701063 genes in environment stress Download PDF

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CN107988234A
CN107988234A CN201711145351.1A CN201711145351A CN107988234A CN 107988234 A CN107988234 A CN 107988234A CN 201711145351 A CN201711145351 A CN 201711145351A CN 107988234 A CN107988234 A CN 107988234A
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CN107988234B (en
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宋纯鹏
李保珠
彭雷
樊若楠
郭竞选
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Henan University
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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Abstract

The invention belongs to gene engineering technology field, and in particular to the patent application that corn GRMZM2G701063 genes are applied in Adversity-stressed Plant reaction is adjusted.The corn GRMZM2G701063 genes, its CDS length are 822bp, encode the R2R3 class myb transcription factors of 273 amino acid.Research shows that the gene is related to corn leaf sheath, tassel growth and development, and compared with wild type B73, after the gene unconventionality expression, corn Phenotypic Expression goes out leaf sheath pastiness（Wild type B73 is embodied in：It is gradually blue under illumination）, tassel it is colourless（Wild type B73 is shown as：Flower pesticide is blue）Phenotypic characteristic, while drought tolerance is remarkably reinforced.Can be that the new variety of plant with particular phenotype cultivates and establishes certain theory and application foundation with preferable stress resistance plant rearing new variety based on these characteristics.

Description

Application of the GRMZM2G701063 genes in environment stress
Technical field
The invention belongs to gene engineering technology field, and in particular to corn GRMZM2G7010633Gene is inverse in adjusting plant The patent application applied in the Stress responses of border.
Background technology
Corn directly affects the survival and development of human society as global first grande culture thing, its yield and quality. And arid is the most important restriction factor of crop yield, at present as one of center topic of Adversity-stressed Plant.
In long-term foliage combat drought analysis, it has been identified that it is many can plant drouhgt stress reaction in play it is important The component of effect, but the research for the identification of these components and their Function approach is mainly to be intended by the thing of pattern The realizations such as southern mustard.And for important crops such as corn, rice, wheat etc., to having drought stress work(in its genome It is relatively fewer that the research of component and can be regulated and controled, thus identify that the important component that drought stress is responded in their genomes and arid are anti- Answer approach that then there is highly important theoretical and realistic meaning.
For corn GRMZM2G701063 genes（Referring to corn biomolecule information database：https:// www.maizegdb.org/gene_center/gene/GRMZM2G701063）, some researches show that：GRMZM2G701063 bases The CDS length of cause is 822bp, encodes the R2R3 class myb transcription factors of 273 amino acid；For with the DNA homolog gene work( Can prediction thinks that the generation of itself and the secondary metabolite of plant has relevance, but do not have and report that it is related to Response to stress.
The content of the invention
The purpose of the application is by the Primary Study to corn GRMZM2G701063 genes, thus to the gene with Function in plant phenotype correlation and Response to stress correlation has preliminary understanding, and then cultivates and established necessarily for New Crop Varieties Theory and application foundation.
Details are as follows for the technical solution that the application is taken.
Application of the corn GRMZM2G701063 genes in plant growth, the corn GRMZM2G701063 genes, its CDS length is 822bp, encodes the R2R3 class myb transcription factors of 273 amino acid, which sends out with corn leaf sheath, tassel growth Correlation is educated, after which causes gene unconventionality expression because of reasons such as fragment deletion, insertion mutations, corn Phenotypic Expression goes out leaf sheath hair It is blue or green（Wild-type corn B73 kinds are embodied in：Corn leaf sheath is gradually blue under illumination condition）, tassel it is colourless（Wild type Corn B73 kinds are embodied in：Flower pesticide is blue）Phenotypic characteristic.
It is a kind of cultivate with particular phenotype feature corn variety method, the particular phenotype feature refer to leaf sheath pastiness, The colourless phenotypic characteristic of tassel, this method is inserted into by gene or gene silent technology loses corn GRMZM2G701063 genes It is living, make its unconventionality expression.
Application of the GRMZM2G701063 genes in environment stress, the gene is related to plant drouhgt stress reaction, the base Because causing exception because of reasons such as deletion mutation, insertion mutations, plant drought tolerance is remarkably reinforced, and the plant is, for example, specifically Corn.
A kind of method for improving plant drought tolerance, this method is inserted into by gene or gene silent technology will GRMZM2G701063 genes or with GRMZM2G701063 DNA homolog gene unconventionalities, make its activity change.
In the prior art, think that the gene may for the gene functional prediction homologous with corn GRMZM2G701063 Certain effect is played in the generation of Secondary metabolites flavonoids（Based on homologous gene functional study）, but on the base The actual functional capability of cause lacks experimental verification always, is lacked in particular for function of the corn GRMZM2G701063 genes in adverse circumstance Weary practical study.
In the application, based on the further investigation of specified mutant material, inventor obtains scarce in Maize mutant storehouse Lose the mutant material of GRMZM2G701063 genetic fragments and and then preliminary analysis research has been done to the function of the gene.Research Show, the gene is related to corn leaf sheath, tassel growth and development, and compared with wild type B73, the gene is because of reasons such as fragment deletions After being abnormal, corn Phenotypic Expression goes out leaf sheath pastiness（Wild type B73 is embodied in：It is gradually blue under illumination）, tassel without Color（Wild type B73 is shown as：Flower pesticide is blue）Phenotypic characteristic, while drought tolerance is remarkably reinforced., can based on these characteristics Cultivated for the new variety of plant with particular phenotype and establish certain theory with preferable stress resistance plant rearing new variety And application foundation.
Brief description of the drawings
Fig. 1 isdos57The screening process of mutant；Wherein A is pairdos57The temperture of leaves detection of mutant（dos57Mutant For infrared temperture of leaves abnormal sudden change body）, B isdos57The temperture of leaves difference quantitative analysis of mutant and corn B73；
Fig. 2 isdos57The phenotypic difference of mutant and corn B73 contrasts；Wherein A for corn B73 anddos57Mutant it is in vitro Leaves water loss compares, and B is grows 10 days on compost, phenotype contrast of the arid after 5 days；C is the fringe type contrast after maturation；
Fig. 3 is to determine mutantdos57Mutation Relevant phenotype by single-gene is recessive control when correlation hybrid experiment；Wherein A is It is returned F1The leaf sheath and drought tolerance phenotype in generation compare, and B is that stalk color and drought tolerance compare figure in hybrid Population；
Fig. 4 is that mutator positioning result figure is carried out from clonal population；
Fig. 5 is sequenced for related gene and gene definitive result；Wherein A is the structure of GRMZM2G701063 genes；B surveys for gene Sequence result（First behavior B73 measures sequence, and lower two behavioral mutants measure sequence）；C is that B73 and mutant measure difference sequence Row peak figure.
Embodiment
The present invention is further explained with reference to embodiment, before specific embodiment is introduced, with regard to following realities Involved part biological material Background situation briefly introduction in example is applied to be described as follows.
Biomaterial：
Maize mutant storehouse：For the mutant library using the natural self-mating system B73 of corn as background material created by EMS mutagenesis, Corn desired phenotype material can therefrom be screened；
Corn B73, a kind of disclosed, most common research corn inbred line；
Corn PH4CV, a kind of disclosed, common research corn inbred line, the first jade 335 cultivated for Pioneer Electronic Corp. of the U.S. The feature such as male parent, its drought tolerance is similar with B73, its genome sequence is not yet announced；
Related sequencing is by raw work bioengineering in the application（Shanghai）Limited company completes.
Embodiment
Due to the correlated results and Maize mutant of the application（dos57Mutant）Acquisition have be directly linked property, because And the present embodiment is first with regard to corndos57The screening of mutant obtains and the clone of corn GRMZM2G701063 genes obtained Journey is briefly discussed below.
First, corndos57The screening of mutant obtains
In greenhouse, cultivation condition is the corn seed cultivation of Maize mutant storehouse：Light dark cycles are 14/10h, and temperature is white My god/at night：30/24 DEG C, relative humidity 70% or so, after seed is sprouted and grows 15 days, observes corn seedling in mutant library Phenotype, while carry out temperture of leaves detection using far infrared imagery instrument.
The result shows that：Mutantdos57Temperture of leaves is abnormal（As shown in Figure 1A）, with its background material（Wild type B73）Temperture of leaves Contrast shows that its temperture of leaves differs by more than 1 degree（As shown in Figure 1B）, there is certain researching value.
2nd, corndos57The phenotype of mutant
Further after inbreeding of more generation, obtain puredos57Mutant seeds.Willdos57Mutant is planted respectively with wild type B73 After plant（Cultivation condition refers to foregoing teachings）, contrast its phenotype, it can be seen that：
（1）dos57Mutant shows the infrared temperture of leaves abnormal phenotype of temperature；
By B73 anddos57It is seeded in same culturing pot at the same time（10cm×10cm）（Nutrition Soil：Vermiculite=1:1）In, it is ensured that soil Water content is between 50-70%；To normally it cultivate 15 days（3 ~ 4 leaf phases）Mutantdos57And B73 seedling carries out excised leaf Dehydration is tested, as a result as shown in Figure 2 A, it can be seen that dos57Mutant percentage of water loss is significant lower；
In Osmotic treatment experiment, to normal culture 10 days or so（To 3 leaf phases）Corn seedling stop watering, carry out Phenotypic Observation And the relevant detection such as infrared；Cutting off the water 5 days or so, B73 anddos57The drought stress phenotype of mutant（Wilt）Occur obvious Difference, for soil moisture content less than 30%, mutant shows stable drought tolerance phenotype at this time（As shown in Figure 2 B）
（2）dos57Mutant seedlings are substantially different from its background material B73 in leaf sheath color and strain tassel（Such as Fig. 2 B and Shown in 2C）；Further, the development phenotype such as its leaf sheath color and strain tassel and drought tolerance phenotype stablize heredity（It was selfed for 7 generations More than）, show that mutator develops phenotype to leaf sheath color and strain tassel etc. and drought tolerance phenotype is related.
3rd, mutantdos57Mutation Relevant phenotype controlled by single-gene is recessive
To determine mutantdos57Mutation Relevant phenotype be by single-gene regulate and control or by polygenes interact regulate and control, hair A person of good sense willdos57Mutant is returned with wild type B73, obtains backcrossing F1, and the seedling of F1 is returned in leaf sheath color It is and identical with B73 in drought tolerance phenotype（As shown in Figure 3A）, show that mutation type surface is controlled by recessive gene.
Again by F1F is produced by stringent selfing2.Qualification result shows, F2, there is phenotype separation, F in generation2Exist for seedling part In stalk color and drought tolerance phenotype with mutantdos57Unanimously（As shown in Figure 3B）.Statistics at the same time, has stalk face Color and the corn seedling of drought tolerance phenotype account for the 1/4 of colony seedling, meet single-gene recessive inheritance, i.e.,：Mutantdos57's Relevant phenotype is mutated by the recessive control of single-gene.
4th, clonal population is built, to mutantdos57The recessive control gene of middle single-gene（Mutator）Positioned
By mutantdos57With another Natural wild-type corn PH4CV（Withdos57Background material refer in drought tolerance etc. Corn inbred line relatively is put on, the map based cloning available for target gene）Carry out the F1 generation of hybridization generation.Further Ground, by F1Carry out stringent selfing and form F2 clonal populations.
From F2There is mutant phenotype for screening in colony（With mutantdos57Phenotype is identical）Clone's single plant.Utilize MaizeGDB websites（https://www.maizegdb.org/）The SSR marker of release, chooses in 10 chromosomes of corn Even distribution possesses codominant SSR marker, and codominance is carried out to the 300 plants of objective trait single plants screened from clonal population SSR marker single plant expands and exchange rate statistics.Heredity rule, the relatively low SSR marker of exchange rate and purpose base are exchanged according to chain It is because more close linkage relationship is presented, i.e., nearer apart from target gene.
Target gene, is tentatively positioned in the range of corn Article 6 chromosome underarm 100-115M by rule more than. More SSR markers are found within the range, utilize greater amount of single plant（4800 or so）Swap rule statistics and single plant Tendency is tested.
Finally, by mutantdos57The recessive control gene of middle single-gene（Mutator）It is positioned at No. six dyeing of corn Between 108.2 ~ 108.6 M of body（As shown in Figure 4）.
5th, for the confirmation of corn GRMZM2G701063 gene mutations
Some researches show that share 9 genes, to determine specifically between 108.2 ~ 108.6 M of No. six chromosome of corn For the character mutation caused by which gene mutation, further examining order is carried out to this 9 genes.
Sequencing result shows that fragment loss phenomenon occurs in only the 3rd of corn GRMZM2G701063 genes extron, and It is clearly single that signal peak is sequenced at the scarce fragment of institute（As shown in Figure 5）.
To further determine that the reliability of result, 10 single plants randomly selected in mutant are carried out to repeat sequencing, are shown Same result is shown.This result shows that, compared with wild type B73, mutantdos57Middle GRMZM2G701063 genes 3rd extron is implicitly present in base and loses and fragment deletion phenomenon, and code segment gene critical function area position.
Now some researches show that：R2R3 type transcription factors in GRMZM2G701063 gene code corns.With corn The research of GRMZM2G701063 genes shows in the arabidopsis of GRMZM2G701063 DNA homologs, in arabidopsis, the gene There are abnormal conditions in terms of thaliana flower pigment after mutation.But specifically effect and and adverse circumstance on the gene in corn Effect in terms of resistance lacks specific verification and research.
In short, having research conditions based on the application can draw：
In corn, corn GRMZM2G701063 genes are related to corn leaf sheath, tassel growth and development, and the gene is because of missing After the reasons such as mutation, insertion mutation cause exception, corn Phenotypic Expression goes out leaf sheath pastiness（The specific table of wild-type corn B73 kinds It is now：Corn leaf sheath is gradually blue under illumination condition）, tassel it is colourless（Wild-type corn B73 kinds are embodied in：Flower pesticide It is blue）Phenotypic characteristic；Based on this characteristic, using related gene engineering technology means, change corn GRMZM2G701063 bases Cause, can cultivate it is new, there is particular phenotype（The colourless phenotype of leaf sheath pastiness, tassel）The corn variety of feature.
And have more application value is then that GRMZM2G701063 genes have correlation with environment stress, correlation is utilized Technique for gene engineering means, by GRMZM2G701063 genes or with after GRMZM2G701063 DNA homolog gene unconventionality expressions, The drought tolerance of plant can be obviously improved, and then for cultivating the new variety of plant with preferable drought tolerance.

Claims (4)

1. application of the corn GRMZM2G701063 genes in plant growth, it is characterised in that the corn GRMZM2G701063 genes, its CDS length are 822bp, encode the R2R3 class myb transcription factors of 273 amino acid, the gene It is related to corn leaf sheath, tassel growth and development；After the gene is abnormal, it is colourless that corn Phenotypic Expression goes out leaf sheath pastiness, tassel Phenotypic characteristic.
2. a kind of cultivate the method with particular phenotype feature corn variety,It is characterized in that,The particular phenotype feature refers to The colourless phenotypic characteristic of leaf sheath pastiness, tassel, this method is inserted into by gene or gene silent technology is by corn GRMZM2G701063 abnormal gene expressions.
3.GRMZM2G701063 application of the gene in environment stress, it is characterised in that the gene is reacted with plant drouhgt stress Correlation, after the gene unconventionality expression, plant drought tolerance is remarkably reinforced.
A kind of 4. method for improving plant drought tolerance, it is characterised in that this method is inserted into by gene or gene silencing Technology by GRMZM2G701063 genes or with GRMZM2G701063 DNA homolog gene unconventionalities.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109666679A (en) * 2019-03-01 2019-04-23 中国农业大学 Chinese rose transcription factor RhPTM and its application
CN112680472A (en) * 2019-10-17 2021-04-20 华南农业大学 Application of ZmSPL gene in regulation and control of development of corn crown root or aerial root
CN112795552A (en) * 2021-03-10 2021-05-14 河南大学 Application of Zm0001d024568 gene and encoding protein thereof in drought stress resistance of corn
CN115700282A (en) * 2022-12-21 2023-02-07 河南大学三亚研究院 Corn drought-resistant gene Zm00001d030678 and application thereof

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CN106755076A (en) * 2016-12-29 2017-05-31 新乡医学院 Applications of the arabidopsis gene COLD1 in terms of Genes For Plant Tolerance drought stress

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109666679A (en) * 2019-03-01 2019-04-23 中国农业大学 Chinese rose transcription factor RhPTM and its application
CN112680472A (en) * 2019-10-17 2021-04-20 华南农业大学 Application of ZmSPL gene in regulation and control of development of corn crown root or aerial root
CN112680472B (en) * 2019-10-17 2023-08-01 华南农业大学 Application of ZmSPL gene in regulation and control of maize crown root or aerial root development
CN112795552A (en) * 2021-03-10 2021-05-14 河南大学 Application of Zm0001d024568 gene and encoding protein thereof in drought stress resistance of corn
CN115700282A (en) * 2022-12-21 2023-02-07 河南大学三亚研究院 Corn drought-resistant gene Zm00001d030678 and application thereof
CN115700282B (en) * 2022-12-21 2023-11-03 河南大学三亚研究院 Maize drought-resistant gene Zm00001d030678 and application thereof

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