CN114250231A

CN114250231A - Low-temperature lethal and high-temperature response rice leaf color mutant gene and detection method and application thereof

Info

Publication number: CN114250231A
Application number: CN202111370022.3A
Authority: CN
Inventors: 陈瑜; 高媛媛; 林冬枝; 董彦君
Original assignee: Shanghai Normal University
Current assignee: Shanghai Normal University
Priority date: 2021-11-18
Filing date: 2021-11-18
Publication date: 2022-03-29

Abstract

The invention discloses a specific primer capable of specifically amplifying low-temperature lethal and high-temperature response rice leaf color mutant gene fragments, and the low-temperature lethal and high-temperature response rice leaf color mutant genes can be detected by comparing sequences obtained by sequencing after amplification with parents.

Description

Low-temperature lethal and high-temperature response rice leaf color mutant gene and detection method and application thereof

Technical Field

The invention relates to the fields of agriculture, plant biotechnology and the like, in particular to the basic research of rice genetic breeding, gene function, physiology and the like at the molecular level.

Technical Field

The leaves are the main organs of plants for photosynthesis, are rich in types and easy to identify, so that the leaf color is used for distinguishing and judging the growth condition of plants and plant mutationOne of the simplest and intuitive simple criteria. In practical application, the leaf color can be used for breeding novel varieties of ornamental plants, can also be used as the most ideal morphological marker of a plurality of crops such as wheat, rice, corn and the like, and can also be used in genetic breeding, physiological and biochemical mechanisms of plants and cloning and function research of related genes thereof. At present, researchers have created a large number of leaf color mutants of rice by using physical, chemical, biological and other techniques, and the leaf color mutants are classified into leaf color mutants of high temperature expression type, low temperature expression type, temperature insensitive type and the like according to whether the leaf color mutants are regulated by temperature. This study utilizes⁶⁰Co gamma-ray mutagenesis treatment is carried out on the created low-temperature lethal and high-temperature response rice leaf color gene mutant strain, and a new gene of the low-temperature lethal and high-temperature response rice leaf color mutant gene is positioned by a map-based cloning technology.

Disclosure of Invention

The invention aims to provide a low-temperature lethal and high-temperature response rice leaf color mutant gene, protein coded by the gene, a detection method and application of the gene.

Rice with low-temperature lethal and high-temperature response to leaf color change comes from the channel⁶⁰The rice character and various agronomic characters are stable after the group progeny generated by the mutagenesis of the japonica rice Jiahua No.1 treated by Co gamma rays is subjected to multiple selfing propagation and selection. F obtained by hybridizing the rice material with indica rice variety through wide-affinity culture of dwarf 64S₂The generation segregation population is used as a positioning population, and the gene of the leaf color of the low-temperature sensitive rice is positioned on the No.3 chromosome of the rice by using a DNA molecular marker.

The rice plant carrying the low-temperature lethal and high-temperature response rice leaf color mutant gene shows whitening under the condition of growth below 20 ℃, and then dies after four leaf periods; albinism was observed at 32 ℃ incubation, and survival continued after a trefoil period with greening. The gene has a nucleotide sequence shown in SEQ ID No. 1. The protein coded by the gene has an amino acid sequence shown in SEQ ID No. 2.

A pair of specific primers for detecting the genes, the sequences of which are as follows:

upstream: 5'-GTTTGAAGAGATGAAGAACAGTG-3'

Downstream: 5'-CTTTTTTATGTCACCTTGCTTGC-3'

Namely SEQ ID No.3 and SEQ ID No.4

The method for detecting the low-temperature lethal and high-temperature response rice leaf color mutant gene comprises the step of adopting the specific primers (SEQ ID No.3 and SEQ ID No. 4) to extract DNA of rice and amplifying to obtain a nucleotide sequence of 409 bp. The sequences of SEQ ID No.5 and SEQ ID No.6 are divided into parents and shown in the low-temperature lethal and high-temperature response rice leaf color mutant gene rice. A basic group G is deleted at 172bp in a 408bp fragment obtained by amplifying rice DNA carrying low-temperature lethal and high-temperature response rice leaf color mutant genes.

In the invention, the inventor designs a specific primer capable of specifically amplifying the low-temperature lethal and high-temperature response rice leaf color mutant gene fragment, and the sequence obtained by sequencing after amplification is compared with the parent sequence, so that the low-temperature lethal and high-temperature response rice leaf color mutant gene can be detected.

Drawings

FIG. 1 shows the phenotype of the mutant rice with low-temperature lethal and high-temperature response rice leaf color at 20 deg.C (left) and 32 deg.C (middle) and wild-type (right) seedling stage;

FIG. 2 shows the result of sequence comparison between the PCR primer amplification product and wild rice according to the present invention.

Detailed Description

Example 1 extraction of DNA from Rice

Taking fresh rice leaves in the seedling stage, cutting the leaves into small sections with the length of 0.5cm, putting the small sections into a precooled mortar, adding liquid nitrogen to grind the small sections into powder, transferring the powder into a 2mL centrifuge tube, adding 1mL of 2% CTAB (cell membrane damage) solution preheated at 60 ℃, slightly shaking the solution, then carrying out water bath at 60 ℃ for 45min, and shaking the solution for multiple times.

Taking out the centrifuge tube, cooling to room temperature, centrifuging at 12000r/min at 20 deg.C for 5min, sucking 600 μ L of supernatant, adding equal volume of chloroform-isoamyl alcohol (24: 1) mixture, and mixing well.

The tube containing the solution was equilibrated, centrifuged at 12000r/min at 20 ℃ for 5min, 600. mu.L of the supernatant was aspirated, and 2/3 volumes (400. mu.L) of isopropanol were added thereto, followed by gentle mixing to precipitate nucleic acids.

12000r/min centrifugation at 20 ℃ for 3min to precipitate nucleic acid, and discard the supernatant.

Adding 70% ethanol for washing, centrifuging at 12000r/min at 20 deg.C for 5min, and discarding the supernatant.

Repeat step 5

Dried at room temperature and dissolved in 150. mu.L of sterile water.

Take 2. mu.L for the subsequent PCR amplification reaction.

Example 2

Obtaining the leaf color mutant gene of rice with low-temperature lethal and high-temperature response and the amino acid sequence thereof

The 25 μ L PCR reaction was as follows: 100mM Tris-HCl pH9.0, 100mM KCl, 20mM MgSO₄;80mM (NH₄)SO₄2.5Mm Dntp, 10 μ M primer, 5U/. mu.L Taq enzyme, 1 μ L template DNA.

The primer sequence is as follows:

upstream: 5'-ggatcttccagagatCTCCTGTTCCCCTCTGCCTG-3'

Downstream: 5'-ctgccgttcgacgatGGAAAAAAAATAGACTGAAGTATAACTCAGA-3'

And (3) carrying out PCR amplification reaction.

The length of the PCR amplification product is 5627bp, a gene sequence obtained by downloading a Bioxm software and http:// rice. plant biology. msu. edu/webpage is compared with a sequencing result, the result of the fragment is shown as SEQ ID No.1, and the result of the amino acid obtained by translating the sequence is shown as SEQ ID No.2 according to the result.

(II) Gene for detecting leaf color of temperature-regulated rice

A1.25 μ L PCR reaction was as follows: 100mM Tris-HCl pH9.0, 100mM KCl, 20mM MgSO₄;80mM (NH₄)SO₄2.5Mm Dntp, 10 μ M primer, 5U/. mu.L Taq enzyme, 1 μ L template DNA.

The specific PCR primer sequences are as follows:

upstream: 5'-GTTTGAAGAGATGAAGAACAGTG-3'

Downstream: 5'-CTTTTTTATGTCACCTTGCTTGC-3'

2. And (3) carrying out PCR amplification reaction.

The resulting amplification product was stained with bromophenol blue, loaded on a 1.5% agarose gel stained with ethidium bromide, electrophoresed, imaged on a UVP gel imager, and a 408bp band appeared and the amplification product was sent for detection. The sequence obtained was compared with the parental sequence using BioXM software, the result is shown in FIG. 2, the sequence is shown in SEQ ID No.6, which is a stretch of SEQ ID No. 1.

Example 4 functional Gene validation

Gene of leaf color mutant rice subjected to low-temperature death and high-temperature response comes from the channel⁶⁰The filial generation of the group generated by the mutagenesis of the japonica rice Jiahua No.1 treated by Co gamma rays is bred and selected by selfing for a plurality of times, and the mutation character and various agronomic characters are stable. The rice plant carrying the gene shows whitening under the condition of cultivation at 20 ℃, and then dies after four-leaf period; the plant shows albinism under the condition of cultivation at 32 ℃, and survives after the trilobate period with greenish recovery, and has obvious characters of low-temperature sensitive death and high-temperature response revival.

Through⁶⁰After Co gamma ray treatment, sequencing results show that the gene has base substitution shown in the base sequence of SEQ ID No. 1.

The nucleotide sequence containing the rice self promoter SEQ ID number 7 is applied to an agrobacterium tumefaciens expression vector pCAMBIA1301, and the target gene sequence is verified through sequencing.

The recombinant vector is transformed into a receptor by utilizing an agrobacterium-mediated technology, the receptor is a rice callus containing low-temperature lethal and high-temperature response rice leaf color mutant genes, a positive first-generation transgenic plant is obtained by identification, the leaf color of the positive first-generation transgenic plant is whitened below 20 ℃, and the positive first-generation transgenic plant is transferred to a condition of 32 ℃ and cultured to generate a greening phenomenon in a three-leaf period.

The seeds of the first generation transgenic plants are sown at low temperature, the leaf color of the seeds is separated at the seedling stage, the seeds have green and albino expression, and the Mendelian genetic segregation law is 3: 1.

The rice carrying the temperature-regulated leaf color gene is green in the four-leaf stage of the leaf color at 32 ℃, and dies after the leaf color of the plant in the seedling stage is whitened at 20 ℃, so that the truth of the rice variety can be identified through the phenotypic change, and the purity of the hybrid rice seed is improved.

Sequence listing

<110> Shanghai university of Master

<120> a low-temperature lethal and high-temperature response rice leaf color mutant gene, and detection method and application thereof

<130> none

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5627

<212> DNA

<213> Rice Sequence (Rice Sequence)

<400> 1

ctcctgttcc cctctgcctg ccttcacgga gaacacgccg ccgcacgccc gcaaagttgt 60

cgctccgccg ccgggtcctg cggccacttc ctccctctcc ctgtgcatgc gctctcttcc 120

ccacctgtac tttactttag ctgctcctct gcccagttgc ccacgacctg acgacccgga 180

catggcgcag gctgaggcgg ggacgacgac atcgccggcg ggttgacgca gaaaggagcg 240

accacccgag ggctccgctg gattttcagg tagctgagct gagctgaact gaaccccaat 300

gccccgcgtt tgcgccgccc ctcgggcgcc gccgccgccg tgcccgtgcc atgtcggagt 360

agggccgctt cggccgaggt ggcgcgcctc ccggcacggc cctctccggg cggctggcca 420

ggagcagctc ctcaccgccc tgcgcgagca gccggacccc gacgcggcgc tccggatgct 480

caacgcggcg ctggcgcggg acgacttcgc gcccggcccc gaggtctacg aggagatcat 540

ccgcaagctc ggcgcggtcg gggccctcga cctcatgaag gtgctcgtcg cggagatgcg 600

gcgggagggg caccaggtga aattgggcgt agtccactcc ttcttggaca gctacgaggg 660

gcagcagctg ttcgacgatg ccgtcgacct gattctgaat caactccaac cattgtttgg 720

cattcaggca gacaccgtgg tgtacaatca ccttctcaat gttcttgtgg aggggagcaa 780

aatgaaactc cttgaatcag tgtactcgga gatgggtgct aggggaatca agcctgatgt 840

tgtcacattc aacacactga tgaaggcgtt gtgccgagca catcaggtca ggactgcagt 900

tctcatgctc gaggaaatgt ctagcagagg cgtggcgcct gacgagacga cgtttaccac 960

cctgatgcaa ggatttgtcg aggaggggag catcgaggct gcactgaggg tcaaagccag 1020

gatgttggaa atggggtgct cggcgacgaa ggtgacggtt aatgttttga ttaatgggta 1080

ctgcaagcta gggagggtgg aggatgctct tgggtatata cagcaggaga ttgccgatgg 1140

gtttgagcct gaccagatca catataacac ttttgttaat ggtctctgcc aaaatgatca 1200

tgtcggccat gccctcaaag tcatggatgt gatggttcag gagggccatg atcctgatgt 1260

tttcacctac aatatcgttg tgaattgcct ttgtaaaaat ggacagcttg aagaggcaaa 1320

aggaattctg aatcagatgg tggatcgggg ttgtttgcct gacattacca cattcaacac 1380

tctcattgct gccttatgca cggggaatcg acttgaggaa gccttggacc ttgcacgtca 1440

ggttacagtg aagggagtct ctccagatgt ttatactttc aatattctga ttaacgcgct 1500

ctgcaaagta ggcgatcctc atcttgcact tcgattgttt gaagagatga agaacagtgg 1560

atgcaccccg gatgaagtaa catacaatac tttgattgac aatctttgct cacttgggaa 1620

gcttggtaaa gcattggatt tgttaaaaga tatggagtcc actggttgtc ctcgaagtac 1680

aattacatat aacactataa ttgacgggtt atgcaagaaa atgagaatag aagaagcaga 1740

agaagttttt gatcaaatgg atctgcaagg catttcgagg aatgcaatca cattcaatac 1800

tctcatcgat ggtttgtgca aggacaaaaa gattgatgat gcttttgagc ttattaatca 1860

aatgataagt gaagggttgc aacctaacaa tatcacttac aattctattc taactcatta 1920

ttgcaagcaa ggtgacataa aaaaggctgc ggatatttta gaaactatga ctgcaaatgg 1980

atttgaagtg gatgttgtta cgtacggtac tctgattaac ggtctatgca aggctggtag 2040

gacacaggtt gctttgaagg tactcagagg tatgcggata aaagggatga ggcctactcc 2100

aaaagcttac aatcctgtgc tccagtctct cttcagacgg aataatatca gagatgccct 2160

gagtcttttc agggagatgg cagaggttgg tgagcctcct gatgctttga catataagat 2220

tgtttttcgt gggctctgtc gtggtggagg gcctattaaa gaagcttttg atttcatgtt 2280

ggagatggtt gataaggggt tcataccaga gttctcatcc ttccgtatgc tagctgaagg 2340

tctattaaac ctgggtatgg atgattactt cattagagcc attgaaataa tcatggaaaa 2400

ggtcgacctc agagagtctg atgtttctgc aataagggga tatctcaaga tccgcaaatt 2460

ttatgatgca ttagcaacct ttggccgttt cctggagatc aacaaccctc aatggagtta 2520

ccgatgaagc agaatacata actgggacaa attacttgaa tagtattagg gaaatctcaa 2580

agagtggatg gaatttttgc tggttgctta ggggaatgaa agtcttaaat tgattataat 2640

aggtgattgt gttcattcct cggtagggat gaagtcagag catgaagaag ctcatcttgg 2700

tgcagaaact tagcttattg gaacagaatc taggtgctag gtgctgtctg ctgccagatt 2760

agtacctagc gccttaacga acagtggctg cagatcccat ccgcttattt tgatcaaatc 2820

tgattcattt tctattccct aataaaagcc tgattcatct tcattgcata tggtcgaacc 2880

taaggctatc atgtacagtt agatcccaac ccttcgttct atgagatgtt gtcccataga 2940

agaaatatct tctgagtata tcctagtact ctaaaatgtt cactaatatg ttcaacttaa 3000

ttagttttgt aacctcctaa aatagttcta ttagttttgt aaccttctaa aatatgaatt 3060

agttttgatc tggctgatct tcctttggtt aggtactaca aattcttaat tcagacacat 3120

atttggtttt ctgaaaattt catctatatt tggtcaggct ggcatttgaa gttcttattt 3180

tagtcatata ctttagtttt ttacaatttt catttgttaa agatgataat ttatttgtta 3240

gcacagagca tgtttagaaa tctgaaatat taaaacatgc atgttctcat gaaaataaat 3300

gttagttttg tttaaattcc aatccacata ttttttaatc aatgtcagaa attaccatgc 3360

ttcacttatt gacctagtat atgtatagta tttgatggat catgttgatt tggatttgct 3420

ctactaactt gttcctattc caacaaagat ttatatgcat cttgtgttct aaaaatgcta 3480

catgtgtcaa gttgaaggaa aattctagct atgtggtgtc ctaattttgg tagatggtac 3540

ctagtaatag ataacaattc tgttttatag tgatgagtaa atttgactaa atcgagtcta 3600

gaaagtgata taattttctg gagaagtctt tcttggtgat ttgggaaagg gccattacct 3660

atactgatat gaaatctgga actagaaaga tctgaacatc aatgttctaa agttttttgt 3720

ctgaatttct tgtgcagaat atgaaggaag gtggatctgg aataggtatt tacatgtcct 3780

gttcagattc ctgcaatctg ataaactact gcaatccaat aaactcttga tctattgttt 3840

tctggatttt tttttgggtg tagagtatta ggaaggtatt tgctttgtta caggggttgg 3900

ttggatgttc agaaaaccaa aatctgaata cactaacaca ttagccagtg ttttatttta 3960

gtttatgtta ttctgaccac aacaccagca gcttcaattg gtagtagaac acactctgat 4020

gcaattggta ggtgtacaac agataatttg ccgtgaatgc tacttaattc aaccattttt 4080

tttccataca gtgctataca atggcacaca aacatccttc agttggactc aaatttgttt 4140

cagctttgct attttacagt cacctgcagt tttttctaat ccatcagtcg attttgttgg 4200

cagtagattc tgcttcaaga tctgtgcacg cagtggaggc acatttggtt acaggctgct 4260

tcaccagacc cgttaaccca tgggccatgg atgctcccct agtagcatat gttgtttttt 4320

ccaccaactg cctcttgtaa atcaagatgc tcccctctcc acatttgctg cagacttcct 4380

ccccgtggat ctgagacgaa ctccggcgac cggcggcgtc tacgagccag cgttcaccaa 4440

tttctcaggt atatgagtcg tcatctatgt gcatctgcta cttttttttt ttggctgatt 4500

ggacgggttg agaaggaggt gtttgggggg aagagagcag aataatccca ttgcgaaaca 4560

agcgaggaag gcttgtcgat ggtggcggcg gtggccttga ttatcgaaga tgcatgcgtg 4620

agggaaactg tgtgccaggg aggcaagctt ggtggcacac atcgagtggt atatcttcag 4680

gttactgcta gatcgaagat cctttggtgc ttgatgtgtg ctgttggttt ggtagttttg 4740

gtggtactgt ggcaagtgaa gacacgaaag aggagctagt agaggcttag agtctggagt 4800

cgtaatcgat gtgcaagagc atcaatgcca tcagttcctc tgggcagcac ggctctgcac 4860

ttttcatctg agaaacatat atattttgtt gattctgatt ttgacgaatt atccaaacat 4920

gcagtttttt tgggttccgc gtgcaaagac gattttgatc tcctggcaga tttaagggtg 4980

gtgattttgt ttttgttttg catgtagatg atgcattttg taaatcattt taggttgccc 5040

cttttttttt tgtttttgcg agttcttact gaatttggat aagttctggt tgattggatc 5100

gacattctgt acatttgcaa ggggattcta tgggtttttg cttattgcta atcaaattat 5160

tttgtgagtt ttggtttatt ggatggtgag aaagcaagat tacatggatt ttgcttcatt 5220

gctgattttg tcgaaattta tcaggcatgc ggtttttggt ttcttgaata cgaactacac 5280

ggacagtact cctatattct tggtgatttt gaagtgtatt tgttattttg gggaggtgga 5340

atggttaata ttttgttcaa ggggacactt ctacagatgt atacattact tttgtcattt 5400

tcagcataac aaagagattt ggtagattca gaattcagat ggcaactaca cgtcgaatga 5460

tgtatgcgaa gacatgggga attctgatgc tgattccact gaagaaagct aaacataatt 5520

ttaatttatt taccaactca tttttctgtc aacacatttg ctagatagtt gctacccgat 5580

aaacgacatc ttgaaatctg agttatactt cagtctattt tttttcc 5627

<210> 2

<211> 742

<212> PRT

<213> amino acid Sequence (PRT Sequence)

<400> 2

Met Pro Arg Val Cys Ala Ala Pro Arg Ala Pro Pro Pro Pro Cys Pro

1 5 10 15

Cys His Val Gly Val Gly Pro Leu Arg Pro Arg Trp Arg Ala Ser Arg

20 25 30

His Gly Pro Leu Arg Ala Ala Gly Gln Glu Gln Leu Leu Thr Ala Leu

35 40 45

Arg Glu Gln Pro Asp Pro Asp Ala Ala Leu Arg Met Leu Asn Ala Ala

50 55 60

Leu Ala Arg Asp Asp Phe Ala Pro Gly Pro Glu Val Tyr Glu Glu Ile

65 70 75 80

Ile Arg Lys Leu Gly Ala Val Gly Ala Leu Asp Leu Met Lys Val Leu

85 90 95

Val Ala Glu Met Arg Arg Glu Gly His Gln Val Lys Leu Gly Val Val

100 105 110

His Ser Phe Leu Asp Ser Tyr Glu Gly Gln Gln Leu Phe Asp Asp Ala

115 120 125

Val Asp Leu Ile Leu Asn Gln Leu Gln Pro Leu Phe Gly Ile Gln Ala

130 135 140

Asp Thr Val Val Tyr Asn His Leu Leu Asn Val Leu Val Glu Gly Ser

145 150 155 160

Lys Met Lys Leu Leu Glu Ser Val Tyr Ser Glu Met Gly Ala Arg Gly

165 170 175

Ile Lys Pro Asp Val Val Thr Phe Asn Thr Leu Met Lys Ala Leu Cys

180 185 190

Arg Ala His Gln Val Arg Thr Ala Val Leu Met Leu Glu Glu Met Ser

195 200 205

Ser Arg Gly Val Ala Pro Asp Glu Thr Thr Phe Thr Thr Leu Met Gln

210 215 220

Gly Phe Val Glu Glu Gly Ser Ile Glu Ala Ala Leu Arg Val Lys Ala

225 230 235 240

Arg Met Leu Glu Met Gly Cys Ser Ala Thr Lys Val Thr Val Asn Val

245 250 255

Leu Ile Asn Gly Tyr Cys Lys Leu Gly Arg Val Glu Asp Ala Leu Gly

260 265 270

Tyr Ile Gln Gln Glu Ile Ala Asp Gly Phe Glu Pro Asp Gln Ile Thr

275 280 285

Tyr Asn Thr Phe Val Asn Gly Leu Cys Gln Asn Asp His Val Gly His

290 295 300

Ala Leu Lys Val Met Asp Val Met Val Gln Glu Gly His Asp Pro Asp

305 310 315 320

Val Phe Thr Tyr Asn Ile Val Val Asn Cys Leu Cys Lys Asn Gly Gln

325 330 335

Leu Glu Glu Ala Lys Gly Ile Leu Asn Gln Met Val Asp Arg Gly Cys

340 345 350

Leu Pro Asp Ile Thr Thr Phe Asn Thr Leu Ile Ala Ala Leu Cys Thr

355 360 365

Gly Asn Arg Leu Glu Glu Ala Leu Asp Leu Ala Arg Gln Val Thr Val

370 375 380

Lys Gly Val Ser Pro Asp Val Tyr Thr Phe Asn Ile Leu Ile Asn Ala

385 390 395 400

Leu Cys Lys Val Gly Asp Pro His Leu Ala Leu Arg Leu Phe Glu Glu

405 410 415

Met Lys Asn Ser Gly Cys Thr Pro Asp Glu Val Thr Tyr Asn Thr Leu

420 425 430

Ile Asp Asn Leu Cys Ser Leu Gly Lys Leu Gly Lys Ala Leu Asp Leu

435 440 445

Leu Lys Asp Met Glu Ser Thr Gly Cys Pro Arg Ser Thr Ile Thr Tyr

450 455 460

Asn Thr Ile Ile Asp Gly Leu Cys Lys Lys Met Arg Ile Glu Glu Ala

465 470 475 480

Glu Glu Val Phe Asp Gln Met Asp Leu Gln Gly Ile Ser Arg Asn Ala

485 490 495

Ile Thr Phe Asn Thr Leu Ile Asp Gly Leu Cys Lys Asp Lys Lys Ile

500 505 510

Asp Asp Ala Phe Glu Leu Ile Asn Gln Met Ile Ser Glu Gly Leu Gln

515 520 525

Pro Asn Asn Ile Thr Tyr Asn Ser Ile Leu Thr His Tyr Cys Lys Gln

530 535 540

Gly Asp Ile Lys Lys Ala Ala Asp Ile Leu Glu Thr Met Thr Ala Asn

545 550 555 560

Gly Phe Glu Val Asp Val Val Thr Tyr Gly Thr Leu Ile Asn Gly Leu

565 570 575

Cys Lys Ala Gly Arg Thr Gln Val Ala Leu Lys Val Leu Arg Gly Met

580 585 590

Arg Ile Lys Gly Met Arg Pro Thr Pro Lys Ala Tyr Asn Pro Val Leu

595 600 605

Gln Ser Leu Phe Arg Arg Asn Asn Ile Arg Asp Ala Leu Ser Leu Phe

610 615 620

Arg Glu Met Ala Glu Val Gly Glu Pro Pro Asp Ala Leu Thr Tyr Lys

625 630 635 640

Ile Val Phe Arg Gly Leu Cys Arg Gly Gly Gly Pro Ile Lys Glu Ala

645 650 655

Phe Asp Phe Met Leu Glu Met Val Asp Lys Gly Phe Ile Pro Glu Phe

660 665 670

Ser Ser Phe Arg Met Leu Ala Glu Gly Leu Leu Asn Leu Gly Met Asp

675 680 685

Asp Tyr Phe Ile Arg Ala Ile Glu Ile Ile Met Glu Lys Val Asp Leu

690 695 700

Arg Glu Ser Asp Val Ser Ala Ile Arg Gly Tyr Leu Lys Ile Arg Lys

705 710 715 720

Phe Tyr Asp Ala Leu Ala Thr Phe Gly Arg Phe Leu Glu Ile Asn Asn

725 730 735

Pro Gln Trp Ser Tyr Arg

740

<210> 3

<211> 23

<212> DNA

<213> primers (Primer Sequence)

<400> 3

gtttgaagag atgaagaaca gtg 23

<210> 4

<211> 23

<212> DNA

<213> primers (Primer Sequence)

<400> 4

cttttttatg tcaccttgct tgc 23

<210> 5

<211> 409

<212> DNA

<213> Wild type Rice (Wild type rice Sequence)

<400> 5

gtttgaagag atgaagaaca gtggatgcac cccggatgaa gtaacataca atactttgat 60

tgacaatctt tgctcacttg ggaagcttgg taaagcattg gatttgttaa aagatatgga 120

gtccactggt tgtcctcgaa gtacaattac atataacact ataattgacg ggttatgcaa 180

gaaaatgaga atagaagaag cagaagaagt ttttgatcaa atggatctgc aaggcatttc 240

gaggaatgca atcacattca atactctcat cgatggtttg tgcaaggaca aaaagattga 300

tgatgctttt gagcttatta atcaaatgat aagtgaaggg ttgcaaccta acaatatcac 360

ttacaattct attctaactc attattgcaa gcaaggtgac ataaaaaag 409

<210> 6

<211> 408

<212> DNA

<213> Mutant Gene (Mutant gene Sequence)

<400> 6

gtttgaagag atgaagaaca gtggatgcac cccggatgaa gtaacataca atactttgat 60

tgacaatctt tgctcacttg ggaagcttgg taaagcattg gatttgttaa aagatatgga 120

gtccactggt tgtcctcgaa gtacaattac atataacact ataattgacg gttatgcaag 180

aaaatgagaa tagaagaagc agaagaagtt tttgatcaaa tggatctgca aggcatttcg 240

aggaatgcaa tcacattcaa tactctcatc gatggtttgt gcaaggacaa aaagattgat 300

gatgcttttg agcttattaa tcaaatgata agtgaagggt tgcaacctaa caatatcact 360

tacaattcta ttctaactca ttattgcaag caaggtgaca taaaaaag 408

<210> 7

<211> 5626

<212> DNA

<213> Agrobacterium expression vector Sequence

<400> 7

ctcctgttcc cctctgcctg ccttcacgga gaacacgccg ccgcacgccc gcaaagttgt 60

cgctccgccg ccgggtcctg cggccacttc ctccctctcc ctgtgcatgc gctctcttcc 120

ccacctgtac tttactttag ctgctcctct gcccagttgc ccacgacctg acgacccgga 180

catggcgcag gctgaggcgg ggacgacgac atcgccggcg ggttgacgca gaaaggagcg 240

accacccgag ggctccgctg gattttcagg tagctgagct gagctgaact gaaccccaat 300

gccccgcgtt tgcgccgccc ctcgggcgcc gccgccgccg tgcccgtgcc atgtcggagt 360

agggccgctt cggccgaggt ggcgcgcctc ccggcacggc cctctccggg cggctggcca 420

ggagcagctc ctcaccgccc tgcgcgagca gccggacccc gacgcggcgc tccggatgct 480

caacgcggcg ctggcgcggg acgacttcgc gcccggcccc gaggtctacg aggagatcat 540

ccgcaagctc ggcgcggtcg gggccctcga cctcatgaag gtgctcgtcg cggagatgcg 600

gcgggagggg caccaggtga aattgggcgt agtccactcc ttcttggaca gctacgaggg 660

gcagcagctg ttcgacgatg ccgtcgacct gattctgaat caactccaac cattgtttgg 720

cattcaggca gacaccgtgg tgtacaatca ccttctcaat gttcttgtgg aggggagcaa 780

aatgaaactc cttgaatcag tgtactcgga gatgggtgct aggggaatca agcctgatgt 840

tgtcacattc aacacactga tgaaggcgtt gtgccgagca catcaggtca ggactgcagt 900

tctcatgctc gaggaaatgt ctagcagagg cgtggcgcct gacgagacga cgtttaccac 960

cctgatgcaa ggatttgtcg aggaggggag catcgaggct gcactgaggg tcaaagccag 1020

gatgttggaa atggggtgct cggcgacgaa ggtgacggtt aatgttttga ttaatgggta 1080

ctgcaagcta gggagggtgg aggatgctct tgggtatata cagcaggaga ttgccgatgg 1140

gtttgagcct gaccagatca catataacac ttttgttaat ggtctctgcc aaaatgatca 1200

tgtcggccat gccctcaaag tcatggatgt gatggttcag gagggccatg atcctgatgt 1260

tttcacctac aatatcgttg tgaattgcct ttgtaaaaat ggacagcttg aagaggcaaa 1320

aggaattctg aatcagatgg tggatcgggg ttgtttgcct gacattacca cattcaacac 1380

tctcattgct gccttatgca cggggaatcg acttgaggaa gccttggacc ttgcacgtca 1440

ggttacagtg aagggagtct ctccagatgt ttatactttc aatattctga ttaacgcgct 1500

ctgcaaagta ggcgatcctc atcttgcact tcgattgttt gaagagatga agaacagtgg 1560

atgcaccccg gatgaagtaa catacaatac tttgattgac aatctttgct cacttgggaa 1620

gcttggtaaa gcattggatt tgttaaaaga tatggagtcc actggttgtc ctcgaagtac 1680

aattacatat aacactataa ttgacggtta tgcaagaaaa tgagaataga agaagcagaa 1740

gaagtttttg atcaaatgga tctgcaaggc atttcgagga atgcaatcac attcaatact 1800

ctcatcgatg gtttgtgcaa ggacaaaaag attgatgatg cttttgagct tattaatcaa 1860

atgataagtg aagggttgca acctaacaat atcacttaca attctattct aactcattat 1920

tgcaagcaag gtgacataaa aaaggctgcg gatattttag aaactatgac tgcaaatgga 1980

tttgaagtgg atgttgttac gtacggtact ctgattaacg gtctatgcaa ggctggtagg 2040

acacaggttg ctttgaaggt actcagaggt atgcggataa aagggatgag gcctactcca 2100

aaagcttaca atcctgtgct ccagtctctc ttcagacgga ataatatcag agatgccctg 2160

agtcttttca gggagatggc agaggttggt gagcctcctg atgctttgac atataagatt 2220

gtttttcgtg ggctctgtcg tggtggaggg cctattaaag aagcttttga tttcatgttg 2280

gagatggttg ataaggggtt cataccagag ttctcatcct tccgtatgct agctgaaggt 2340

ctattaaacc tgggtatgga tgattacttc attagagcca ttgaaataat catggaaaag 2400

gtcgacctca gagagtctga tgtttctgca ataaggggat atctcaagat ccgcaaattt 2460

tatgatgcat tagcaacctt tggccgtttc ctggagatca acaaccctca atggagttac 2520

cgatgaagca gaatacataa ctgggacaaa ttacttgaat agtattaggg aaatctcaaa 2580

gagtggatgg aatttttgct ggttgcttag gggaatgaaa gtcttaaatt gattataata 2640

ggtgattgtg ttcattcctc ggtagggatg aagtcagagc atgaagaagc tcatcttggt 2700

gcagaaactt agcttattgg aacagaatct aggtgctagg tgctgtctgc tgccagatta 2760

gtacctagcg ccttaacgaa cagtggctgc agatcccatc cgcttatttt gatcaaatct 2820

gattcatttt ctattcccta ataaaagcct gattcatctt cattgcatat ggtcgaacct 2880

aaggctatca tgtacagtta gatcccaacc cttcgttcta tgagatgttg tcccatagaa 2940

gaaatatctt ctgagtatat cctagtactc taaaatgttc actaatatgt tcaacttaat 3000

tagttttgta acctcctaaa atagttctat tagttttgta accttctaaa atatgaatta 3060

gttttgatct ggctgatctt cctttggtta ggtactacaa attcttaatt cagacacata 3120

tttggttttc tgaaaatttc atctatattt ggtcaggctg gcatttgaag ttcttatttt 3180

agtcatatac tttagttttt tacaattttc atttgttaaa gatgataatt tatttgttag 3240

cacagagcat gtttagaaat ctgaaatatt aaaacatgca tgttctcatg aaaataaatg 3300

ttagttttgt ttaaattcca atccacatat tttttaatca atgtcagaaa ttaccatgct 3360

tcacttattg acctagtata tgtatagtat ttgatggatc atgttgattt ggatttgctc 3420

tactaacttg ttcctattcc aacaaagatt tatatgcatc ttgtgttcta aaaatgctac 3480

atgtgtcaag ttgaaggaaa attctagcta tgtggtgtcc taattttggt agatggtacc 3540

tagtaataga taacaattct gttttatagt gatgagtaaa tttgactaaa tcgagtctag 3600

aaagtgatat aattttctgg agaagtcttt cttggtgatt tgggaaaggg ccattaccta 3660

tactgatatg aaatctggaa ctagaaagat ctgaacatca atgttctaaa gttttttgtc 3720

tgaatttctt gtgcagaata tgaaggaagg tggatctgga ataggtattt acatgtcctg 3780

ttcagattcc tgcaatctga taaactactg caatccaata aactcttgat ctattgtttt 3840

ctggattttt ttttgggtgt agagtattag gaaggtattt gctttgttac aggggttggt 3900

tggatgttca gaaaaccaaa atctgaatac actaacacat tagccagtgt tttattttag 3960

tttatgttat tctgaccaca acaccagcag cttcaattgg tagtagaaca cactctgatg 4020

caattggtag gtgtacaaca gataatttgc cgtgaatgct acttaattca accatttttt 4080

ttccatacag tgctatacaa tggcacacaa acatccttca gttggactca aatttgtttc 4140

agctttgcta ttttacagtc acctgcagtt ttttctaatc catcagtcga ttttgttggc 4200

agtagattct gcttcaagat ctgtgcacgc agtggaggca catttggtta caggctgctt 4260

caccagaccc gttaacccat gggccatgga tgctccccta gtagcatatg ttgttttttc 4320

caccaactgc ctcttgtaaa tcaagatgct cccctctcca catttgctgc agacttcctc 4380

cccgtggatc tgagacgaac tccggcgacc ggcggcgtct acgagccagc gttcaccaat 4440

ttctcaggta tatgagtcgt catctatgtg catctgctac tttttttttt tggctgattg 4500

gacgggttga gaaggaggtg tttgggggga agagagcaga ataatcccat tgcgaaacaa 4560

gcgaggaagg cttgtcgatg gtggcggcgg tggccttgat tatcgaagat gcatgcgtga 4620

gggaaactgt gtgccaggga ggcaagcttg gtggcacaca tcgagtggta tatcttcagg 4680

ttactgctag atcgaagatc ctttggtgct tgatgtgtgc tgttggtttg gtagttttgg 4740

tggtactgtg gcaagtgaag acacgaaaga ggagctagta gaggcttaga gtctggagtc 4800

gtaatcgatg tgcaagagca tcaatgccat cagttcctct gggcagcacg gctctgcact 4860

tttcatctga gaaacatata tattttgttg attctgattt tgacgaatta tccaaacatg 4920

cagttttttt gggttccgcg tgcaaagacg attttgatct cctggcagat ttaagggtgg 4980

tgattttgtt tttgttttgc atgtagatga tgcattttgt aaatcatttt aggttgcccc 5040

tttttttttt gtttttgcga gttcttactg aatttggata agttctggtt gattggatcg 5100

acattctgta catttgcaag gggattctat gggtttttgc ttattgctaa tcaaattatt 5160

ttgtgagttt tggtttattg gatggtgaga aagcaagatt acatggattt tgcttcattg 5220

ctgattttgt cgaaatttat caggcatgcg gtttttggtt tcttgaatac gaactacacg 5280

gacagtactc ctatattctt ggtgattttg aagtgtattt gttattttgg ggaggtggaa 5340

tggttaatat tttgttcaag gggacacttc tacagatgta tacattactt ttgtcatttt 5400

cagcataaca aagagatttg gtagattcag aattcagatg gcaactacac gtcgaatgat 5460

gtatgcgaag acatggggaa ttctgatgct gattccactg aagaaagcta aacataattt 5520

taatttattt accaactcat ttttctgtca acacatttgc tagatagttg ctacccgata 5580

aacgacatct tgaaatctga gttatacttc agtctatttt ttttcc 5626

Claims

1. The low-temperature lethal and high-temperature response rice leaf color mutant gene is characterized by having a nucleotide sequence shown in SEQ ID No. 1.

2. The low temperature lethal and high temperature response rice leaf color mutant gene as claimed in claim 1, wherein the nucleotide sequence thereof is shown in SEQ ID No. 1.

3. The protein encoded by the gene sequence of claim 1 or 2, having 742 amino acid sequences shown in SEQ ID No. 2.

4. The protein of claim 3, wherein the amino acid sequence is set forth in SEQ ID No. 2.

5. A pair of specific PCR primers for detecting the low-temperature lethal and high-temperature responsive rice leaf color mutant genes according to claim 1 or 2, wherein the nucleotide sequences thereof are:

upstream: 5'-GTTTGAAGAGATGAAGAACAGTG-3' (SEQ ID No. 3)

Downstream: 5'-CTTTTTTATGTCACCTTGCTTGC-3' (SEQ ID No. 4).

6. The method for detecting the low-temperature lethal and high-temperature response rice leaf color mutant genes as claimed in claim 1 or 2, which is characterized in that the specific PCR primer as claimed in claim 5 is used for carrying out PCR amplification reaction on DNA extracted from rice seedling stage to obtain nucleotide sequences of 409bp as shown in SEQ ID No.5 (wild type rice) and SEQ ID number 6 (carrying the low-temperature lethal and high-temperature response rice leaf color mutant genes).

7. The detection method as claimed in claim 6, wherein the 408bp fragment obtained by amplifying the DNA of the rice plant carrying the low temperature lethal and high temperature responsive leaf color mutant gene has a change of one base G deletion compared with the 409bp fragment obtained by amplifying the DNA of the parent, indicating that the leaf color gene of the low temperature sensitive rice is contained.