CN103173431B

CN103173431B - Applications of insertion of Arabidopis thaliana heat shock protein gene HSP101 and its T-DNA to mutant strain hot1

Info

Publication number: CN103173431B
Application number: CN201310076606.9A
Authority: CN
Inventors: 李唯奇; 禹晓梅; 陶发清; 郁步竹
Original assignee: Kunming Institute of Botany of CAS
Current assignee: Kunming Institute of Botany of CAS
Priority date: 2012-01-16
Filing date: 2012-01-16
Publication date: 2014-08-06
Anticipated expiration: 2032-01-16
Also published as: CN103173431A

Abstract

The invention provides applications of the insertion of an Arabidopis thaliana heat shock protein gene HSP101 and its T-DNA to a mutant strain hot1 in the change of seed germination and preservation, comprising an application of the insertion of the gene T-DNA to the mutant strain hot1 in transgenic plants for changing the seed germination and preservation, and an application in the cultivation of seeds having a low abscisic acid (ABA) content, a high gibberellin (GA3) content and a high indoleacetic acid (IAA) content. In the invention, a case that the Arabidopis thaliana heat shock protein gene HSP101 in a mode plant Arabidopis thaliana has seed germination and ageing related functions, and the deletion of the gene makes Arabidopis thaliana seeds be sensitive to artificial ageing and not resist storage in the natural preservation process, the endogenous hormone ABA is reduced, the GA3 is risen, and the IAA is risen, and it is in favor of germination. The operation gene HSP101 can change the seed preservation and germination characteristics, and has a good application prospect in the genetic resource preservation and crop production.

Description

The application of Arabidopis thaliana heat shock protein gene HSP101 and T-DNA insertion mutation strain hot1 thereof

The application is the divisional application of patent application " application of Arabidopis thaliana heat shock protein gene HSP101 in seed germination and preservation " (application number: 2012100123017, the applying date: on January 16th, 2012).

Technical field:

The invention belongs to seed physiology and plant genetic engineering field.Particularly, relate to arabidopsis gene HSP101 and T-DNA insertion mutation strain hot1 thereof in the application changing in seed preservation, Germination Characteristics, also relate to the application in the transgenic plant of this gene T-DNA insertion mutation strain hot1 aspect changing seed germination and preservation simultaneously, and there is low levels dormin (ABA) in cultivation, high-content Plant hormones regulators,gibberellins (GA ₃) and the seed of growth hormone (IAA) in application.

Background technology:

The preservation of seed is the important means of Preservation of plant germplasin, its protection to species diversity, agriculture production is had great importance.According to statistics, the plant germplasm resource of having collected in the world has 6,100,000 parts, wherein 90% is kept in low temperature Germplasm Bank with seed form, and the germplasm quantity of the long-term storage of China has reached 340,000 parts, occupies first place in the world.But germplasm cryopreservation also exists a lot of problems, what wherein Genetic Integrity of Germplasm Resource is had the greatest impact is exactly Seed Aging problem, shows as percentage of germination and reduces.Seed germinating power in naturally storing also can be lost gradually, weather aging that Here it is (Machado Neto, Custodio et al.2001).Weather aging lasts longer, is difficult to application in breed improvement while carrying out germplasm screening; Temper(ing) can overcome the deficiency that under normal temperature, weather aging required time is grown, and is widely used in the research of Seed Storability, and temper(ing) often adopts hot and humid method.In the process of Seed Aging, can there is a series of Physiology and biochemistry and heredity variation; comprise: the variation of variation, organic content and kind and the variation of genetic construction (Galleschi, the Capocchi et al.2002 of plasma membrane injury, material energy metabolism; Andreev, Spiridonova et al.2004; Freitas, Dias et al.2006).Therefore, carry out the correlative study of Seed Aging, disclose Mechanism of Seed Aging and further find out relevant countermeasure, preserve and have important practical significance for germ plasm resource.

Seed is the important production means in agricultural, horticultural production, and it sprouts the production that directly affects agricultural gardening.People manage to make the neat recovery growth rapidly of embryo breaking dormancy for sowing obtains neat seedling shape seedling, for high yield lays the foundation.The sprouting of seed is subject to the coordinated regulation (Bewley1997) of environmental factors (as light, temperature and humidity etc.) and internal factor (as color and the structure etc. of gene, hormone, sugar, nitrogenous compound, seed coat).Hormone regulating and controlling aspect, Plant hormones regulators,gibberellins (GA ₃), growth hormone (IAA) and dormin (ABA) be the main plant hormone of regulation and control seed germination and dormancy.ABA suppresses to sprout, induced dormancy, and Plant hormones regulators,gibberellins and growth hormone promote to sprout, and are the Main Factors of antagonism ABA, are seed germination institute essential (Flores, Jurado et al.2006; Rizza, Boccaccini et al.2010).At present, not yet clear and definite to " switch " mechanism of sprouting by dormancy, the especially research aspect to Regulation in Seed Germination genes involved.

Heat shock protein Heat Shock Protein(HSP) be extensively present in (Zhang in the tissue such as plant cell membrane, tenuigenin, chloroplast(id), plastosome, Wang et al.2007), one group of protein that structure is conservative, according to the homology of its molecular size range and sequence, can be divided into five classes (Papp, Nardai et al.2003) such as small HSPs (sHSPs), HSP60, HSP70, HSP90 and HSP100.Their major function has: (1) prevents the protein aggregation of heated denaturalization; (2) by the denatured protein of having assembled separately; (3) protein folding of auxiliary sex change returns its original configuration; (4) assist to decompose strained protein (Suk-Whan and Elizabeth2001).Most heat shock protein is high conservative on developing.In Antisense Suppression mode, stop the expression of Arabidopis thaliana HSP101, can make mutant strain lose acquired thermotolerance, mutant strain, still cannot be at the environment existence (Gurley2000 of 45 DEG C under the pre-treatment of 90 minutes through 38 DEG C; Suk-Whan and Elizabeth2001).Heat shock protein and thermal excited transcryption factor except working under heat shock, also relevant with other multiple abiotic stress, as arid, and high salt and low temperature stress etc. (Kotak, Larkindale et al.2007).Tomato sHSP can be by low temperature induction, and HSP21 is found to be included in anti-oxidant approach, and At HSP17.6 can be induced by osmotic pressure.At seed germination, particularly early stage, genetic transcription is active, and protein is synthetic in a large number, and the propagation of cell and differentiation are very violent, and the environment of cell is in continuous variation, and cell stimulates very responsive to external world.The HSPs in this period changes and effect shows very outstandingly (Hsu, Lai et al.2010).At present, the T-DNA insertion mutation strain hot1 that has no arabidopsis gene HSP101 disappearance and this gene in prior art can change the report of seed preservation, Germination Characteristics.

Summary of the invention:

The object of the present invention is to provide a kind of Arabidopis thaliana heat shock protein gene HSP101 and T-DNA insertion mutation strain hot1 thereof the application in seed germination, preservation and farm crop produce.

The present invention also aims to provide the application in a kind of arabidopsis gene HSP101 and the transgenic plant of T-DNA insertion mutation strain hot1 aspect cultivation has raising seed germination and preservation thereof, and cultivation has low levels dormin (ABA), high-content Plant hormones regulators,gibberellins (GA ₃) and the seed of growth hormone (IAA) in application.

In order to realize above-mentioned purpose of the present invention, the present invention has adopted following technical scheme:

The application of Arabidopis thaliana heat shock protein gene HSP101 in seed germination.

The application of Arabidopis thaliana heat shock protein gene HSP101 in seed storage.

Arabidopis thaliana heat shock protein gene HSP101 has low levels dormin ABA in cultivation, high-content Plant hormones regulators,gibberellins GA ₃with the application in the seed of growth hormone IAA.

Arabidopis thaliana heat shock protein gene HSP101 disappearance makes Arabidopis thaliana seed have higher vigor.

Arabidopis thaliana heat shock protein gene HSP101 disappearance makes Arabidopis thaliana seed to temper(ing) sensitivity, storage endurance not in natural preservation process.

Arabidopis thaliana heat shock protein gene HSP101 disappearance reduces endogenous hormones dormin ABA in Arabidopis thaliana seed, Plant hormones regulators,gibberellins GA ₃raise, growth hormone IAA raises, and is conducive to sprout.

The application of the T-DNA insertion mutation strain hot1 of arabidopsis gene HSP101 in the transgenic plant of cultivating improved seed sprouting.

The application of the T-DNA insertion mutation strain hot1 of arabidopsis gene HSP101 in the transgenic plant of cultivating improved seed storage.

The T-DNA insertion mutation strain hot1 of Arabidopis thaliana HSP101 has low levels dormin ABA in cultivation, high-content Plant hormones regulators,gibberellins GA ₃with the application in the seed of growth hormone IAA.

The numbering of gene HSP101 of the present invention in GenBank is AT1G74310, the long 2736bp of CDS of this gene, and 911 amino acid of encoding, its nucleotide sequence and aminoacid sequence are as shown in table 1.Utilize the Arabidopsis Mutants hot1 of the HSP101 gene T-DNA insertion obtaining from Arabidopis thaliana Biological resources center ABRC (Arabidopsis Biological Resource Center) as research object, the seed of mutant strain is numbered SALK_066374, one section of T-DNA that mutant hot1 comes from carrier pROK2 inserts in the exon of gene HSP101, cause transgenation, the flanking sequence at insertion point place is as shown in table 2.

The present invention finds the sprouting of heat shock protein gene HSP101 function and seed and aging relevant in model plant Arabidopis thaliana, this genetically deficient can make Arabidopis thaliana seed have higher vigor, but to temper(ing) sensitivity, storage endurance not in natural preservation process, endogenous hormones dormin (ABA) reduces simultaneously, Plant hormones regulators,gibberellins (GA ₃) raise, growth hormone (IAA) raises, and is conducive to sprout.Therefore the present invention finds to operate gene HSP101 and can change seed preservation and Germination characteristics, germ plasm resource preserve and farm crop production in have good application prospect.

Brief description of the drawings:

Fig. 1 is Western Blot qualification;

Fig. 2 is PCR test, and Fig. 2 A is PCR design of primers schematic diagram; Fig. 2 B is electrophoresis result theory expectation schematic diagram;

Fig. 3 is PCR qualification electrophoresis result;

Fig. 4 is Seed Germination of Arabidopsis Pumila gesture;

Fig. 5 is the weather aging of Arabidopis thaliana seed, and Fig. 5 A is Arabidopis thaliana seed weather aging 2 years; Fig. 5 B is 2 years germination rates of Arabidopis thaliana seed weather aging;

Fig. 6 is the temper(ing) of Arabidopis thaliana seed;

Fig. 7 is Arabidopis thaliana Seed Endogenous Hormones content; Fig. 7 A is seed endogenous GA 3 content; Fig. 7 B is seed Endogenous IAA content; Fig. 7 C is seed endogenous aba content.

Embodiment 1:

The acquisition of mutant strain:

The numbering of Arabidopis thaliana heat shock protein gene HSP101 of the present invention in GenBank is AT1G74310, the long 2736bp of CDS of this gene, nucleotide sequence is (table 1) as shown in sequence table, 911 amino acid of encoding, and aminoacid sequence is (table 1) as shown in sequence table.The T-DNA that has bought this gene from U.S.'s Arabidopis thaliana Biological resources center Arabidopsis Biological Resource Center (ABRC) inserts strain seed (SALK_066374), mutant strain name is called hot1, and its insertion point flanking sequence is as table 2.

CDS sequence and the aminoacid sequence of table 1HSP101

1 ATGAATCCAGAGAAATTCACACACAAGACAAACGAGACAATTGCTACAGCTCATGAGCTA

1 METAsnProGluLysPheThrHisLysThrAsnGluThrIleAlaThrAlaHisGluLeu

61 GCTGTGAATGCAGGACATGCTCAATTCACTCCTTTGCATTTAGCTGGTGCTTTGATCTCT

21 AlaValAsnAlaGlyHisAlaGlnPheThrProLeuHisLeuAlaGlyAlaLeuIleSer

121 GATCCCACCGGTATATTTCCTCAAGCAATCTCTAGTGCCGGTGGCGAGAACGCAGCTCAA

41 AspProThrGlyIlePheProGlnAlaIleSerSerAlaGlyGlyGluAsnAlaAlaGln

181 TCTGCTGAAAGAGTGATCAATCAAGCCTTGAAGAAGCTTCCTTCACAATCTCCTCCACCT

61 SerAlaGluArgValIleAsnGlnAlaLeuLysLysLeuProSerGlnSerProProPro

241 GATGATATTCCAGCGAGTTCTAGTCTTATTAAGGTCATTCGTCGTGCTCAAGCTGCTCAG

81 AspAspIleProAlaSerSerSerLeuIleLysValIleArgArgAlaGlnAlaAlaGln

301 AAGTCACGAGGTGATACTCATTTGGCTGTTGACCAGTTGATTATGGGTCTTCTTGAAGAT

101 LysSerArgGlyAspThrHisLeuAlaValAspGlnLeuIleMETGlyLeuLeuGluAsp

361 TCTCAAATCAGGGATTTGTTGAACGAAGTCGGTGTAGCGACGGCGAGGGTAAAGTCTGAG

121 SerGlnIleArgAspLeuLeuAsnGluValGlyValAlaThrAlaArgValLysSerGlu

421 GTTGAGAAGCTTCGTGGGAAAGAAGGGAAGAAAGTTGAGAGTGCTTCAGGGGACACAAAT

141 ValGluLysLeuArgGlyLysGluGlyLysLysValGluSerAlaSerGlyAspThrAsn

481 TTTCAAGCTTTAAAGACTTATGGAAGAGATTTGGTTGAGCAAGCAGGGAAGCTTGATCCT

161 PheGlnAlaLeuLysThrTyrGlyArgAspLeuValGluGlnAlaGlyLysLeuAspPro

541 GTGATTGGTCGTGATGAGGAGATTAGAAGAGTCGTGAGGATTCTTTCGAGGAGAACGAAG

181 ValIleGlyArgAspGluGluIleArgArgValValArgIleLeuSerArgArgThrLys

601 AACAATCCTGTGCTATTGGAGAGCCAGGAGTTGGTAAAACAGCTGTGGTTGAAGGTTTA

201 AsnAsnProValLeuIleGlyGluProGlyValGlyLysThrAlaValValGluGlyLeu

661 GCACAAAGGATTGTGAAAGGAGATGTGCCCAACAGTCTTACTGATGTGAGATTAATTTCG

221 AlaGlnArgIleValLysGlyAspValProAsnSerLeuThrAspValArgLeuIleSer

721 TTGGACATGGGTGCGTTAGTTGCTGGTGCTAAATACCGAGGAGAGTTTGAAGAAAGGTTG

241 LeuAspMETGlyAlaLeuValAlaGlyAlaLysTyrArgGlyGluPheGluGluArgLeu

781 AAATCTGTTTTGAAAGAAGTTGAGGACGCTGAAGGCAAAGTGATTCTCTTTATTGATGAG

261 LysSerValLeuLysGluValGluAspAlaGluGlyLysValIleLeuPheIleAspGlu

841 ATTCATTTGGTTCTTGGTGCTGGCAAAACTGAAGGGTCGATGGATGCAGCTAATCTGTTC

281 IleHisLeuValLeuGlyAlaGlyLysThrGluGlySerMETAspAlaAlaAsnLeuPhe

901 AAGCCCATGTTAGCTAGAGGGCAGCTTCGATGCATTGGTGCTACAACGCTTGAAGAATAC

301 LysProMETLeuAlaArgGlyGlnLeuArgCysIleGlyAlaThrThrLeuGluGluTyr

961 AGGAAATATGTTGAGAAAGATGCTGCCTTTGAGAGGAGGTTCCAACAAGTCTATGTTGCG

321 ArgLysTyrValGluLysAspAlaAlaPheGluArgArgPheGlnGlnValTyrValAla

1021 GAGCCAAGTGTGCCTGACACCATTAGTATCCTTAGAGGACTCAAGGAGAAGTATGAGGGA

341 GluProSerValProAspThrIleSerIleLeuArgGlyLeuLysGluLysTyrGluGly

1081 CATCATGGTGTGCGAATCCAAGACAGAGCTCTTATAAATGCTGCTCAGCTGTCTGCTCGT

361 HisHisGlyValArgIleGlnAspArgAlaLeuIleAsnAlaAlaGlnLeuSerAlaArg

1141 TACATAACTGGTCGGCATTTACCGGATAAAGCAATTGATTTGGTTGATGAGGCTTGTGCG

381 TyrIleThrGlyArgHisLeuProAspLysAlaIleAspLeuValAspGluAlaCysAla

1201 AATGTGAGAGTCCAGCTTGATAGTCAACCTGAAGAGATTGATAACCTTGAAAGGAAGAGG

401 AsnValArgValGlnLeuAspSerGlnProGluGluIleAspAsnLeuGluArgLysArg

1261 ATGCAGCTGGAAATTGAACTTCACGCCTTGGAAAGGGAGAAGGATAAAGCCAGCAAAGCT

421 METGlnLeuGluIleGluLeuHisAlaLeuGluArgGluLysAspLysAlaSerLysAla

1321 CGACTTATAGAGGTGCGGAAAGAGCTTGATGACCTGAGAGACAAGCTTCAGCCTCTCACG

441 ArgLeuIleGluValArgLysGluLeuAspAspLeuArgAspLysLeuGlnProLeuThr

1381 ATGAAATACAGAAAGGAGAAAGAGAGAATTGATGAGATTCGAAGGCTTAAACAGAAAAGA

461 METLysTyrArgLysGluLysGluArgIleAspGluIleArgArgLeuLysGlnLysArg

1441 GAAGAGCTCATGTTTTCTTTGCAGGAGGCAGAACGAAGATATGACCTTGCAAGAGCTGCT

481 GluGluLeuMETPheSerLeuGlnGluAlaGluArgArgTyrAspLeuAlaArgAlaAla

1501 GATCTAAGATATGGCGCAATTCAAGAAGTGGAATCTGCAATTGCCCAACTTGAAGGAACT

501 AspLeuArgTyrGlyAlaIleGlnGluValGluSerAlaIleAlaGlnLeuGluGlyThr

1561 TCTTCTGAAGAGAATGTGATGCTCACAGAAAACGTTGGGCCTGAACACATTGCTGAGGTT

521 SerSerGluGluAsnValMETLeuThrGluAsnValGlyProGluHisIleAlaGluVal

1621 GTGAGCCGTTGGACAGGGATTCCAGTGACGAGACTTGGCCAAAATGAGAAGGAGAGGTTG

541 ValSerArgTrpThrGlyIleProValThrArgLeuGlyGlnAsnGluLysGluArgLeu

1681 ATTGGTCTTGCTGATAGGTTGCATAAGCGGGTTGTGGGACAGAATCAAGCGGTAAATGCA

561 IleGlyLeuAlaAspArgLeuHisLysArgValValGlyGlnAsnGlnAlaValAsnAla

1741 GTTTCTGAGGCAATTCTAAGGTCAAGGGCAGGACTTGGAAGGCCACAACAGCCAACTGGA

581 ValSerGluAlaIleLeuArgSerArgAlaGlyLeuGlyArgProGlnGlnProThrGly

1801 TCATTCTTATTCCTTGGACCAACTGGTGTTGGCAAAACTGAGCTCGCCAAGGCTCTTGCT

601 SerPheLeuPheLeuGlyProThrGlyValGlyLysThrGluLeuAlaLysAlaLeuAla

1861 GAGCAGCTGTTTGATGATGAAAACCTCTTAGTTCGGATTGATATGTCGGAATATATGGAA

621 GluGlnLeuPheAspAspGluAsnLeuLeuValArgIleAspMETSerGluTyrMETGlu

1921 CAACACTCTGTCTCTCGCCTCATTGGGGCACCACCAGGGTATGTTGGTCACGAGGAAGGT

641 GlnHisSerValSerArgLeuIleGlyAlaProProGlyTyrValGlyHisGluGluGly

1981 GGACAACTAACTGAGGCTGTGAGGAGGCGACCTTATTGTGTCATACTCTTTGATGAAGTG

661 GlyGlnLeuThrGluAlaValArgArgArgProTyrCysValIleLeuPheAspGluVal

2041 GAGAAGGCTCATGTTGCTGTCTTCAACACTCTGCTCCAAGTTTTGGATGATGGTCGATTG

681 GluLysAlaHisValAlaValPheAsnThrLeuLeuGlnValLeuAspAspGlyArgLeu

2101 ACAGACGGGCAAGGCAGGACAGTCGATTTCAGGAACTCGGTGATAATCATGACATCAAAC

701 ThrAspGlyGlnGlyArgThrValAspPheArgAsnSerValIleIleMETThrSerAsn

2161 CTTGGTGCTGAACACCTCCTTGCAGGGCTAACTGGGAAAGTAACAATGGAAGTGGCCCGG

721 LeuGlyAlaGluHisLeuLeuAlaGlyLeuThrGlyLysValThrMETGluValAlaArg

2221 GACTGTGTGATGCGGGAGGTGAGGAAACACTTCAGACCAGAGCTCTTGAACAGGCTTGAC

741 AspCysValMETArgGluValArgLysHisPheArgProGluLeuLeuAsnArgLeuAsp

2281 GAGATTGTGGTGTTCGACCCCCTTTCACATGACCAGTTGAGGAAAGTAGCTCGGCTTCAA

761 GluIleValValPheAspProLeuSerHisAspGlnLeuArgLysValAlaArgLeuGln

2341 ATGAAAGACGTTGCTGTCCGGCTTGCTGAAAGAGGAGTTGCTTTGGCAGTCACTGATGCT

781 METLysAspValAlaValArgLeuAlaGluArgGlyValAlaLeuAlaValThrAspAla

2401 GCTTTGGACTATATCTTGGCAGAGAGTTATGACCCGGTGTATGGTGCTAGGCCTATAAGG

801 AlaLeuAspTyrIleLeuAlaGluSerTyrAspProValTyrGlyAlaArgProIleArg

2461 AGATGGATGGAGAAGAAGGTGGTGACAGAACTGTCAAAGATGGTTGTGCGTGAGGAAATC

821 ArgTrpMETGluLysLysValValThrGluLeuSerLysMETValValArgGluGluIle

2521 GATGAAAACTCCACTGTTTACATAGATGCAGGCGCTGGTGATCTTGTGTACCGGGTAGAA

841 AspGluAsnSerThrValTyrIleAspAlaGlyAlaGlyAspLeuValTyrArgValGlu

2581 AGTGGAGGTCTAGTGGACGCTTCAACAGGCAAGAAGTCAGATGTGCTGATTCATATTGCT

861 SerGlyGlyLeuValAspAlaSerThrGlyLysLysSerAspValLeuIleHisIleAla

2641 AACGGGCCAAAGAGAAGTGATGCAGCTCAGGCGGTGAAGAAGATGAGGATCGAGGAAATA

881 AsnGlyProLysArgSerAspAlaAlaGlnAlaValLysLysMETArgIleGluGluIle

2701 GAAGATGACGATAATGAGGAAATGATCGAGGATTAA

901 GluAspAspAspAsnGluGluMETIleGluAsp***

Table 2 insertion point flanking sequence

1ATCTC TGATC CCACC GGTAT ATTTC CTCAA GCAAT CTCTA GTGCC GGTGG

51CGAGA ACGCA GCTCA ATCTG CTGAA AGAGT GATCA ATCAA GCCTT GAAGA

101AGCT(Length:104)

Embodiment 2:

The inspection of mutant strain and homozygote qualification:

Western Blot qualification: wild-type Col and mutant hot1 seed extract total protein, detects the expression of albumen HSP101 with Western Blot.As shown in Figure 1, in wild type seeds, there is positive findings in hsp101 gene normal expression to result, HSP101 albumen can be detected; And there is negative findings in mutant hot1, HSP101 albumen can not be detected, illustrate that in mutant hot1, this gene has been knocked really, can not express.

PCR identifies that homozygote: T-DNA inserts the genotypic qualification of strain and needs three primers, respectively: LP (5 '-AAT AAT GCG GCA AAA GAG GAG-3 '), is positioned at T-DNA insertion point left arm end place on genome; RP (5 '-CTG CTT GCT CAA AAT CTC-3 '), is positioned at T-DNA insertion point right arm end place; Primer BP:LBb3:(5 '-ATTTTGCCGATTTCGGAAC-3 on T-DNA left arm end) .PCR design of primers is with reference to SIGNA (http://signa1.salk.edu/tdnaprimers.2.htm1) related data .PCR principle as shown in Figure 2.

Milling and extracting wild-type Col and mutant hot1 leaves genomic DNA carry out PCR as template.As shown in Figure 3, in the time that this carries out pcr amplification and carry out electrophoresis primer with LP and RP, produce a PCR product band in wild-type plant Col, molecular weight is approximately 1100bp, and in mutant hot1 plant, does not have PCR product to form; In the time that this increases to primer PCR with RP and LBb3, in wild-type plant, there is no PCR product, and in mutant hot1 plant, have a PCR product band.Therefore identify that mutant hot1 plant is homozygote homozygote(HM).

Embodiment 3:

In Arabidopis thaliana, after hsp101 gene knockout, there is higher vigor:

Wild-type Col and mutant hot1 seed are placed in 25 DEG C of illumination of culture dish and sprout, undertaken by standard germination test method, four repetitions are established in each processing, isometric with radicle length and seed, it is germination standard that radicel length reaches seed one half, within 6 days, calculates germination rate.As shown in Figure 4, wild-type Col seed is at 24h, 48h germination rate lower than mutant hot1, and mutant hot1 seed has higher vigor.

Embodiment 4:

In Arabidopis thaliana hsp101 gene be knocked rear naturally storage in vigor lose faster:

Seed is under the condition that storage or Germplasm Bank are preserved naturally, and its germinating power can be lost gradually, and weather aging occurs.This experiment is got the wild-type Col that receives in August, 2009 and mutant hot1 seed storage at room temperature, gets the seed of having stored 2 years in September, 2011 and sprouts the detection vigor of testing.Be placed in 25 DEG C of illumination of culture dish and germinate, in culture dish, use filter paper moisturizing.Carry out 15 days statistics germination rates by standard germination test method.As shown in Figure 5, there is decline in various degree through the weather aging wild-type Col of 2 years and mutant hot1 seed germination rate, wild-type Col germination rate can also reach 85%, and mutant hot1 seed germination rate has dropped to 60%, in Arabidopis thaliana hsp101 gene be knocked in rear weather aging process vigor lose faster, more not resistance to storage.

Embodiment 5:

In Arabidopis thaliana, hsp101 gene is more responsive in temper(ing) process after being knocked:

It is 45 DEG C that wild-type Col and mutant hot1 seed are placed on to temperature, in the environment of relative humidity 100%, and treatment time 0h, 12h, 24h, 48h, 72h.The taking-up seed that is disposed, is placed in and germinates with 25 DEG C of illumination of culture dish of filter paper moisturizing.Carry out 15 days statistics germination rates by standard germination test method.As shown in Figure 6, along with the prolongation of hot and humid ageing treatment time, wild-type Col and mutant hot1 seed are all on a declining curve; Mutant hot1 seed is more responsive, and vigor declines sooner, and burin-in process 24h starts, and along with the prolongation of digestion time, germinating energy declines rapidly.When 48h, germination rate is almost 0%.

Embodiment 6:

In wild-type Col and mutant hot1 seed, endogenous hormones detects:

One, the extraction of sample:

Take the methyl alcohol that 0.2g wild-type Col and mutant hot1 seed add 2ml sample extracting solution 80%, in ice bath, grind to form homogenate, proceed to 10ml test tube, then mortar is rinsed well with the gradation of 2ml extracting solution, proceed in the lump in test tube, after shaking up, be placed in 4 DEG C of refrigerators.4 DEG C are extracted 4h, and the centrifugal 8min of 3500 turn/min, gets supernatant liquor.In precipitation, add 1ml extracting solution, stir evenly, put at 4 DEG C and extract again 1h, centrifugal, merge supernatant liquor recording volume, residue claims dry weight.Supernatant liquor is proceeded in 15ml glass centrifuge tube, dry up with nitrogen, remove the methyl alcohol in extracting solution, use sample liquid constant volume.

Two, sample determination (enzyme-linked immunoassay ELISA)

(1) coated: in the coated damping fluid of 10ml, to add a certain amount of envelope antigen to mix, in the each hole of enzyme plate, add 100uL.Then enzyme plate is bandaged to 3h at 37 DEG C with preservative film.

(2) wash plate: by the plate place to go being coated with, put at room temperature balance.Then get rid of coating buffer.Add washings, place 0.5min and get rid of again washings.Repeat 3 this.

(3) competition: add standard substance, treat test sample and antibody, 37 DEG C of left and right 0.5h.

(4) wash plate: reaction solution is dried, add washings and wash plate four times.

(5) adding two resists: put 37 DEG C of incubation 0.5h.

(6) wash plate: reaction solution is dried, add washings and wash plate four times.

(7) add substrate nitrite ion: take 10-20mg O-Phenylene Diamine (OPD) and be dissolved in 10ml substrate buffer solution, after dissolving completely, add 2-4ul30% hydrogen peroxide.Mix, in every hole, add 100ul, then put into wet box, when after colour developing suitably, every hole adds 50ul2mol/L sulfuric acid termination reaction.

(8) colorimetric: the OD value at the each concentration of sequentially determining standard substance and each sample 490nm place on enzyme linked immunological spectrophotometer.

Comparison of computational results, as shown in Figure 7, in Arabidopis thaliana, hsp101 gene is knocked Plant hormones regulators,gibberellins (GA in rear seed ₃) and the rising of growth hormone (IAA) content, and dormin (ABA) content reduces, and is conducive to sprout.

Claims

1. Arabidopis thaliana heat shock protein gene HSP101 and T-DNA insertion mutation strain hotl thereof have low levels dormin ABA in cultivation, high-content Plant hormones regulators,gibberellins GA ₃with the application in the seed of growth hormone IAA, it is characterized in that Arabidopis thaliana heat shock protein gene HSP101 disappearance reduces endogenous hormones dormin ABA in Arabidopis thaliana seed, Plant hormones regulators,gibberellins GA ₃raise, growth hormone IAA raises, and is conducive to sprout; The numbering of described gene HSP101 in GenBank is AT1G74310, and one section of T-DNA that described mutant strain hotl comes from carrier pROK2 inserts in the exon of gene HSP101, and the seed of mutant hotl strain is numbered SALK_066374.