CN100465275C - Plant salt resistant gene - Google Patents

Abstract

The invention relates to a plant salt tolerance gene sequence in the gene engineering technique field, wherein the gene length is 725bp, open reading frame work encodes 166 amino acids from the 26th -526th nucleic acid. The research that the gene expression is intimidated by 1. 7% after natrium chloratum after 24 hours with semi-quantitative PCR finds that expression quantity of the gene (plant salt tolerance gene) increases to more than 5 times. The clone of the gene increased the gene resources of plant salt tolerance research, which improves more abundant fine candidate gene.

Description

A kind of plant salt tolerance gene

Technical field

The present invention is a kind of plant salt tolerance gene of alternanthera philoxeroides, belongs to gene engineering technology field.

Background technology

At present, the salt tolerance that applying transgene technique improves plant has become common recognition, but isolating plant salt tolerance gene kind and quantity are also very limited, and is mostly that the clone obtains from the salt sensitive plant.The alternanthera philoxeroides salt resistance ability is very strong (referring to people such as David J.Longstreth., vegetable cell, tissue and organ culture, 78:225-230 (2004).), the strong gene of a series of salt resistance abilities should be arranged in its body, be the good material of carrying out resistant gene of salt research.

But since paddy rice and alternanthera philoxeroides all normal growth in fresh water environment, with come from the salt sensitive plant especially the resistant gene of salt of terrestrial plant compare, will change paddy rice over to from the resistant gene of salt of alternanthera philoxeroides may be more effective.Therefore the resistant gene of salt that separates alternanthera philoxeroides for the utilization genetic engineering technique improves especially paddy rice salt tolerance of plant, provides abundant more good candidate gene.

Summary of the invention

The present invention has cloned a kind of plant salt tolerance gene of alternanthera philoxeroides.This mrna length is 725bp, and open reading frame is from Nucleotide 26-526 position, 166 amino acid of encoding.

Embodiment

Adopt sandy culture by pouring Hoagland nutritive medium the indoor cultivation alternanthera philoxeroides, 25 ℃ of 30 ℃ of evenings on daytime, relative air humidity 70%, illumination 12h, light intensity is 400umol/m ²/ s ¹When growing the fibrous root of about 5cm length on the stipes of alternanthera philoxeroides, applying analytical pure sodium-chlor makes its final concentration reach 1.7%, clip fibrous root after 24 hours extracts fibrous root RNA according to INVITROGEN Trizol specification sheets, removes remaining DNA among the RNA according to TAKARA DNaseI specification sheets again.Carry out reverse transcription with the total RNA of 5 μ g according to the M-MLV Reverse Transcriptase specification sheets of Promega company and obtain cDNA.According to nucleotide sequence disclosed by the invention (SEQ ID N0.1), design upstream primer 5 '-GATCAAGTCCCCCACCTA-3 ' and downstream primer 5 '-CGGCAATGATTCAAATATATT-3 ' carries out pcr amplification.The PCR reaction system is as follows: 5ul10 * buffer, 4ul dNTP (2.5mmol.L ^-1), 1ul upstream primer (10 μ mol.L ^-1), 1ul downstream primer (10 μ mol.L ^-1), 0.25ul (5U.ul ^-1) the Taq enzyme, 2ulcDNA replenishes aseptic ddH ₂O to 50ul.The PCR reaction conditions is as follows, 94 ℃ of pre-sex change 1min, 94 ℃ of 30sec, 54 ℃ of 30sec, 72 ℃ of 1min, 35 circulations, 72 ℃ of 10min.After the PCR product being carried out glue recovery purifying, connect the T carrier and transform order-checking.

Detect it by the sxemiquantitative round pcr and handle the back expression at 1.7% sodium-chlor.Vegetable material is cultivated the same, and just material is divided into two portions, is respectively applied for contrast and salt and handles.In order to eliminate the influence of cDNA concentration, select for use house-keeping gene actin to contrast as confidential reference items.The amplification method of Actin is that the cDNA before and after handling with salt is a template, with upstream primer 5 '-TGAACTTCGTGTTGCTCCAGA-3 ' and downstream primer 5 '-AACCCTCGTAGATTGGCACAG-3 ' carries out pcr amplification.The PCR reaction system is as follows: 5ul10 * buffer, 4ul dNTP (2.5mmol.L-1), 1ul upstream primer (10 μ mol.L ^-1), 1ul downstream primer (10 μ mol.L ^-1), 0.25ul (5U.ul ^-1) the Taq enzyme, 2ulcDNA replenishes aseptic ddH ₂O to 50ul.PCR response procedures: 94 ℃ of 3min; 94 ℃ of 20sec, 55 ℃ of 20sec, 72 ℃ of 20sec, 72 ℃ of 10min, the amplified production size is 230bp.Again according to nucleotide sequence disclosed by the invention (SEQ ID N0.1), design upstream primer 5 '-TCGCCCTCATCCCACCAT-3 ' and downstream primer 5 '-GCAGTCACCTGGGTTCTTCG-3 ', the cDNA before and after handling with salt is that template is carried out pcr amplification.The PCR reaction system is as follows: 5ul10 * buffer, 4ul dNTP (2.5mmol.L ^-1), 1ul upstream primer (10 μ mol.L ^-1), 1ul downstream primer (10 μ mol.L ^-1), 0.25ul (5U.ul ^-1) the Taq enzyme, 2ulcDNA replenishes aseptic ddH ₂O to 50ul.PCR response procedures: 94 ℃ of 3min, 94 ℃ of 10sec, 59 ℃ of 10sec, 72 ℃ of 10sec, 72 ℃ of 10min.The amplified production size is 102bp.For pcr amplification was carried out in exponential phase, actin and above-mentioned resistant gene of salt all carry out 25,30,35,40 round-robin amplifications.Index amplification phase PCR product is carried out electrophoresis, then handle fibrous root front and back actin and the brightness of above-mentioned resistant gene of salt PCR product band with gel imaging system observation analysis 1.7% sodium-chlor.After salt was handled, resistant gene of salt was expressed and is strengthened more than 5 times.

Submit the resistant gene of salt sequence that obtains to Genbank, show that through the Blast comparison this gene and pythium spp secreted protein gene homology do not have homology up to 95% with other species.This type of gene function does not appear in the newspapers.Our this resistant gene of salt of above-mentioned evidence expression amount behind salt stress increases by 500 above, is a kind of new resistant gene of salt.The clone of this gene has enlarged the genetic resources of plant salt tolerance research, for the utilization genetic engineering technique improves especially paddy rice salt tolerance of plant, provides abundant more good candidate gene.

＜110〉Central China University of Science and Technology

＜120〉a kind of plant salt tolerance gene

<140>2006101249835

<141>2006—11—09

<160>1

<170>PatentIn?Version?3.3

<210>1

<211>725

<212>DNA

＜213〉alternanthera philoxeroides (Alternanthera philoxeroides)

<400>1

gatcaagtcc?cccacctaca?gcacaatgta?cgccaagacc?ctcctcgtcg?ccgccgtggc 60

cgccctcgct?gccgtcaacg?ccgccccatg?tgacatgctc?actgaggtca?ccaagctgac 120

ccctctcatc?tcggacccaa?acgttgctaa?gtgcagcacc?caggaggagt?cgggcggctt 180

cgccctcatc?ccaccatcgg?gcctgccaac?ccctgaccag?tacaagaaga?tgtgtgtcag 240

cgacgcctgt?aagaaggtca?ttgaggctgt?tgcctcgaag?aacccaggtg?actgcgacct 300

caccgtcggc?tcggtcaccc?tcaacgtgaa?gcagcttgtg?tcgaacttcc?caacggagtg 360

cgccaagtac?acgacgccag?ccaccacggc?tccagcccca?accacgactg?cgccagctcc 420

atcgtcgggc?ccagccccat?cgtcgtcggc?tccagcccca?accggtcaga?ccaccccagc 480

cccaactggc?cagagcaccc?ctgccccaac?caaggctcag?tgctaagtta?tttcgcatct 540

gactcgcggt?cgatggttac?tgctgctcac?tgagtagtgg?tgtcggccca?gtactctcat 600

cgccttcagc?gttttgtcag?tgtccgcgaa?tcaggagctc?atgagcgtcg?ggaggtggtc 660

tatttccctt?cggtcctgct?gaaccgtggt?cgtgtgctgg?aatatatttg?aatcattgcc 720

gtcac 725

Claims (1)

1, a kind of plant salt tolerance gene, its base sequence is shown in SEQ ID NO.1.