CN113151322B - Tobacco starch synthase gene and application thereof - Google Patents

Tobacco starch synthase gene and application thereof Download PDF

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CN113151322B
CN113151322B CN202110574080.1A CN202110574080A CN113151322B CN 113151322 B CN113151322 B CN 113151322B CN 202110574080 A CN202110574080 A CN 202110574080A CN 113151322 B CN113151322 B CN 113151322B
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tobacco
leu
glu
synthase gene
ser
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CN113151322A (en
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曾婉俐
翟妞
向海英
蒋佳芮
李雪梅
高茜
邓乐乐
李晶
杨光宇
杨文武
张建铎
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China Tobacco Yunnan Industrial Co Ltd
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01021—Starch synthase (2.4.1.21)

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Abstract

The application relates to a tobacco starch synthase gene and application thereof, and the base sequence is specifically shown in SEQ ID NO. 1. In the present application, preliminary studies on tobacco starch synthase NtGLGA have found that it is associated with anabolism of starch in tobacco leaves. The gene is silenced in the Nicotiana benthamiana, the content of amylopectin and total starch in tobacco leaves is obviously reduced, the content of the amylopectin is reduced by 64.8%, and the content of the total starch is reduced by 30.2%. Based on the characteristic of tobacco starch synthase NtGLGA, a reference can be provided for tobacco quality regulation and cultivation of new tobacco varieties through genetic engineering breeding.

Description

Tobacco starch synthase gene and application thereof
Technical Field
The application belongs to the technical field of tobacco genetic engineering, and particularly relates to a tobacco starch synthase gene and application thereof.
Background
Starch is the most important basic organic compound in tobacco leaves, and the starch content in mature fresh tobacco leaves is up to about 40%, so that the intrinsic quality and the appearance commodity grade of the tobacco leaves are determined. Starch content is one of the key factors in evaluating tobacco quality. The high starch content of fresh tobacco is unfavorable to quality, especially to the color and flavor of tobacco leaves; the appearance and internal quality of the cured tobacco leaves are affected despite the low starch content. When the sugar existing in the form of starch is burnt and sucked in cigarettes, on one hand, the quality of the smoke is adversely affected, and on the other hand, the burning speed and the completeness are affected. In addition, starch can produce a burnt odor upon combustion, destroying the flavor that is formed upon combustion of tobacco. The starch content (mass fraction) in the flue-cured tobacco in China is about 4% -6%, and the starch content of the foreign high-quality flue-cured tobacco is only 1% -2%.
Therefore, the research on the gene function affecting the starch content in the tobacco provides theoretical support for improving the quality of the tobacco and the genetic improvement of tobacco varieties, and has important significance for improving the quality of tobacco products in China.
Disclosure of Invention
The application aims to provide a tobacco starch synthase gene and application thereof, so as to solve the problem of too high starch content in the existing flue-cured tobacco, and lay a foundation for quality regulation of tobacco leaves and cultivation of new tobacco varieties.
In order to achieve the above purpose, the application is realized by the following technical scheme:
a tobacco starch synthase gene has a base sequence shown in SEQ ID NO.1, contains 3636 bases and is named NtGLGA.
Further, the amino acid sequence of the tobacco starch synthase gene is shown as SEQ ID NO.2, and consists of 1211 amino acid residues.
Further, the PCR amplification preparation method of the tobacco starch synthase gene comprises the following steps:
(1) Extracting genome and reverse transcribing the genome into cDNA for standby;
(2) Designing a primer for PCR amplification, and carrying out PCR amplification, wherein the specific primer sequence is designed as follows:
NtGLGA-F：5’-TTGGAGTTTCTTCTGCAAGGTG-3’，
NtGLGA-R：5’-CAATTACAGGGTTTCCAGACAC-3’。
further, when the genome is extracted in the step (1), leaves of the tobacco variety safflower Dajinyuan are taken as samples.
The use of the tobacco starch synthase gene of any of the above, wherein the protein expressed by the gene is related to the starch content in plant leaves, and after the protein expression is reduced, the amylopectin and total starch content in the leaves is obviously reduced.
Furthermore, the starch content in tobacco leaves is regulated and controlled by regulating the expression level of tobacco starch synthase by using a gene silencing technology or a gene overexpression method.
Further, through a transgenic technology, a transient expression technology or a genome editing technology, a virus-induced silencing vector, an RNAi interference vector and an overexpression vector containing a tobacco starch synthase gene are constructed, tobacco is transformed, and a new tobacco variety with variable starch content is obtained through screening.
Specific examples are: by utilizing the virus-induced gene silencing (VIGS) technology, the expression of the tobacco starch synthase gene is interfered to silence, the amylopectin and total starch content in tobacco starch synthase gene silencing plants is obviously reduced, and then a new plant variety with reduced starch content is obtained.
The beneficial effects of the application are as follows:
in the application, through preliminary research on a specific tobacco starch synthase gene, the gene is found to be highly related to the starch content of tobacco leaves, and the gene is silenced in Nicotiana benthamiana, so that the starch content in the leaves is obviously reduced. Based on the characteristics, a certain application foundation and reference can be provided for tobacco quality regulation and new variety cultivation.
Drawings
FIG. 1 is a graph showing the relative expression of a tobacco starch synthase gene in a silenced plant as compared to a control plant;
FIG. 2 is a comparison of starch content in virus-induced gene-silenced tobacco leaves and control tobacco leaves.
Detailed Description
The following examples are given by way of illustration only and are not to be construed as limiting the scope of the application.
Biological material:
the Nicotiana benthamiana is a common tobacco material at present, seedlings are grown in a seedling pot, seedlings are separated two weeks after germination, and cultivation management such as daily fertilizer and water management is carried out in a plastic pot (10 cm multiplied by 10 cm) at 22 ℃ under 16h light/8 h dark condition.
The VIGS vector used in the examples below is a viral vector (tobacco rattle virus, TRV) from tobacco brittle virus, and the particular TRV2 used (a commonly used vector) carries the kanamycin selectable marker and the 35S promoter, while TRV2 carries multiple cloning sites such as EcoR I and BamH I, which can be used to carry and transform foreign genes.
Experimental reagent:
LB liquid medium, 1L content contains: 10g bactopeptone (bacteriological peptone); 10g of sodium chloride (NaCl); 5g of yeast extract (yeast extract), and sterilizing at high temperature and high pressure.
YEB liquid medium, 1L content contains: 5g beef extract (beef extract); 5g bactopeptone (bacteriological peptone); 5g sucrose (sucrose); 1g of yeast extract (yeast extract); 2mL of 1M magnesium sulfate (MgSO 4) was sterilized at high temperature under high pressure.
1m 2- (N-morpholino) ethanesulfonic acid (MES) stock solution: ddH 2 O is dissolved, filtered and sterilized, and stored at the temperature of minus 20 ℃ for standby.
200mM Acetosyringone (Acetosyringone, as) stock: dimethyl Sulfoxide (DSMO) is dissolved, and stored at-20deg.C for use.
Example 1
This example is briefly described below in terms of the construction of a tobacco NtGLGA gene cloning and silencing vector.
(1) Tobacco NtGLGA gene cloning
According to early-stage researches on tobacco genome and related NtGLGA genes, a specific coding sequence is selected as a target fragment, and a primer sequence for PCR amplification is designed as follows:
NtGLGA-F：5’-TTGGAGTTTCTTCTGCAAGGTG-3’，
NtGLGA-R：5’-CAATTACAGGGTTTCCAGACAC-3’。
the cDNA of the tobacco safflower big golden leaf (firstly extracting genome and then reverse transcribing into cDNA) is used as a template to carry out PCR amplification to obtain the NtGLGA gene.
The PCR amplification procedure was: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 53℃for 15s, extension at 72℃for 3min, and after 34 cycles, extension at 72℃for 5min.
And (3) detecting the PCR amplified product by agarose gel electrophoresis, and recovering the electrophoresis product for standby.
(2) Construction of recombinant TRV2-NtGLGA vector
And (3) carrying out EcoRI and BamHI double digestion on the PCR amplified product in the step (1), simultaneously carrying out EcoRI and BamHI double digestion on the empty vector TRV2, respectively recovering digestion products, and carrying out ligation by using T4 DNA ligase.
The ligation product was transformed into E.coli competent DH 5. Alpha. And after the transformation procedure was completed, the transformation product was spread on LB solid medium containing 50mg/L Kan and incubated overnight at 37 ℃.
And (3) selecting positive single colony for amplification, further carrying out PCR identification, and combining with sequencing verification to ensure that the recombinant vector TRV2-NtGLGA with correct construction is obtained.
The tobacco NtGLGA gene comprises 3636 bases, and the base sequence is shown in SEQ ID NO. 1.
The tobacco starch synthase protein NtGLGA comprises 1211 amino acids, and the amino acid sequence is shown in SEQ ID NO. 2.
Example 2
Based on example 1, the constructed recombinant TRV2-NtGLGA vector was further transformed into tobacco plants using Agrobacterium-mediated VIGS technology, and verification analysis was performed on the phenotype change of the related plants, and the detailed experimental procedure is outlined below.
(1) Transformation of Agrobacterium
It should be noted that, in reference to the procedure of example 1 and the prior art, a TRV2-GFP recombinant vector was prepared as a control, and the specific transformation procedure was as follows:
positive cloning plasmids of TRV2-GFP (vector control) and TRV2-NtGLGA are respectively transformed into competent cells of agrobacterium GV3101 by a shock transformation mode, and are subjected to culture screening by using a YEB plate containing 50mg/L Kan and 50mg/L Rif, and after inversion culture for 2 days at 28 ℃, agrobacterium with a target gene is screened by using colony PCR.
(2) Preparation of bacterial liquid for transfection
The positive Agrobacterium clones selected in step (1) were cultured overnight at 28℃and 250rpm in 5mL of YEB liquid medium (containing 50mg/L Kan and 50mg/L Rif).
Inoculating 50uL of overnight culture into 50mL of YEB liquid culture medium (containing 50mg/L Kan), and culturing to OD 600 =about 1.0-1.5, then centrifuging 4000g for 5min, collecting thallus, re-suspending with MMA, and adjusting OD 600 =about 1.0.
Finally, the cells are left at room temperature for about 3 hours to be used as bacterial liquid for transfection.
(3) Transient transformation
Taking 3-4w (week) of young Nicotiana benthamiana leaves as an experimental material, injecting the bacterial liquid for transfection prepared in the step (2) into the Nicotiana leaves by using a 1mL specification injector, and continuously culturing the injected Nicotiana tabacum leaves in an artificial incubator to observe the phenotype change.
Further, the expression of the NtGLGA gene was detected by qRT-PCR, and the results are shown in fig. 1, and it can be seen that the expression level of NtGLGA is significantly reduced in the infected plant of TRV2-NtGLGA, and the qRT-PCR primer is as follows:
NtGLGA-F：5’-ACAGGCCAACCTTCCTTTG-3’，
NtGLGA-R：5’-GCAGAACCAAGCAAGACCA-3’。
further, the leaf starch content in the experimental group (TRV 2-NtGLGA-infected plants) and the control group (TRV 2-GFP-infected plants) was examined (the examination method was referred to as a branched-chain-amylose-total starch content kit (spectrophotometry) of Grignard organism), and the results are shown in FIG. 2.
As can be seen from the results of FIG. 2, the amylopectin content and the total starch content in the experimental group were significantly reduced compared with the control group, in which the amylopectin content was reduced by 64.2% and the total starch content was reduced by 30.2%. The method further shows that the content of starch in tobacco leaves can be regulated and controlled by silencing the NtGLGA gene, so that a certain technical foundation can be laid for regulating and controlling the quality of tobacco leaves and cultivating new varieties of tobacco.
The foregoing has shown and described the basic principles, principal features and advantages of the application. It will be understood by those skilled in the art that the present application is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present application, and various changes and modifications may be made without departing from the spirit and scope of the application, which is defined in the appended claims. The scope of the application is defined by the appended claims and equivalents thereof.
Sequence listing
<110> Yunnan Zhongyan industry Limited liability company
<120> a tobacco starch synthase gene and use thereof
<130> WPC211438
<141> 2021-05-25
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tccgtacaat cttcttcgtg gaggaacgat ggaatggttg ctggagtttc atatccattt 180
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agggaccgca agtctcagga ggaggaggat gaattcaatg gtgccactaa atcagtaagt 480
ctatcacctg ttcgtggatc aactcaattt gttggaagtg gagaaactgg tgacaatgac 540
gtgggtgctg taaattttaa taaatcaaat agaacggaag agagtgattt tcaaattgat 600
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ggttataaag ttgaacaata caatcatgat gagttggatg agacacaaaa actgaaagaa 780
aatgatgctg ggaatgttga agacaaaaga cccttagcaa gagagctgtt ggaaatgact 840
aagcctagca atgtggaatt tactgaaagc aatgagatca ctgaagtaga cagtaatagt 900
ttcttaaaac cagattcagt tgacgagagt gagccatcaa ctgtaggaac attagagact 960
gaagatattt ctctgaagtt aagattggag atggaagcga atctacgtag gcaggctata 1020
aaaaggcttg ccgaggaaaa tttattgcaa ggaatcagat tattttgttt cccagaggtt 1080
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aaggtcaagt tatactatga caaaagttca ggtcctctct cccatgctaa ggacttgtgg 1680
atccatggag gatataacaa ctggaaggat ggtttgtcta tcgttgaaaa gcttgttaaa 1740
tctgagagaa tagatggtga ttggtggtat acagaggttg tcatccctga tcgggcactt 1800
gtcttggatt gggtttttgc tgatggtcca cccaagcatg ccattgcata tgataacaat 1860
catcgccaag acttccatgc cattgtcccc aagcacattc cggaggaatt atattgggtt 1920
gaggaagaac ttcagatctt caaggcactt caggaggaga gaaggcttag agaagaggct 1980
atgcgtgcta aggctgaaaa agcagcacgt atgaaagctg aaacaaagga aaggactatg 2040
aaatcatttt tattgtctca gaagcacata gtgtacactg agcctcttga tgtccaagct 2100
ggaagcagcg tcacagttta ctataatccc gccaatacag tacttaatgg taaacctgaa 2160
atttggttca gatgttcatt caatcgttgg actcaccgcc tgggtccatt gccgcctcaa 2220
aaaatgttgc ctactgaaaa tggaacccat gtcaaagcaa ctgtgaaggt tccattggat 2280
gcacatatga tggattttgt attctctgag agagaagatg gtggaatttt tgacaataga 2340
agtggaatgg actatcacat acctgtgttt ggaggagtcg caaaagaacc tccgatgcat 2400
attgttcata ttgctgtcga aatggcacct attgcaaagg tgggaggcct tggtgatgtt 2460
gttactagtc tttcccgtgc agttcaagat ttaaaccata atgtggatat catcttacct 2520
aagtatgact gtttgaagat gaatcaggtg aaggactttc agtttcacaa aagctacttt 2580
tggggtggga ctgaaataaa agtatggttt gggaaggtgg aaggtgtctc cgtctacttt 2640
ttggagcctc aaaacgggtt attttggaag gggtgtgtat atggttgtaa taatgatggt 2700
gaaagatttg gtttcttttg tcacgcggct ttggagtttc ttctgcaagg tggatttcat 2760
ccggatatca tccattgcca tgattggtct agtgctcccg ttgcgtggct ctttaaggaa 2820
caatatacac actatggtct aagcaagtct cgaattgtct tcacaataca caatcttgaa 2880
tttggtgcag atctcattgg gaaagcaatg acttatgcag acaaagctac aacagtttcc 2940
ccaacttact caagggaggt gtctggaaac cctgtaattg cacctcacct ttacaagttc 3000
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ccgatttcat acacctcgga aaatgttgtt gaaggcaaaa cagcagccaa ggaagctttg 3120
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aaaactggag gactttatga tactgtattt gatgttgacc atgacaaaga gagagcacaa 3540
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Lys Asp Val Tyr Asp Asn Asn Asp Asn Asn Asp Phe Ser Ile Thr Val
435 440 445
Glu Gly Gly Met Gln Ile Leu Asp Phe Glu Asn Phe Leu Leu Glu Glu
450 455 460
Lys Arg Arg Glu Gln Glu Lys Leu Ala Lys Glu Gln Ala Glu Arg Glu
465 470 475 480
Arg Leu Ala Glu Glu Gln Arg Arg Ile Glu Ala Glu Lys Ala Ala Leu
485 490 495
Glu Ala Asp Arg Ser Gln Ala Lys Glu Glu Ala Ala Lys Lys Arg Glu
500 505 510
Val Leu Gln Ala Leu Met Ala Lys Ala Ser Lys Thr Arg Asp Ile Thr
515 520 525
Trp Tyr Ile Glu Pro Ser Val Phe Lys Cys Glu Glu Lys Val Lys Leu
530 535 540
Tyr Tyr Asp Lys Ser Ser Gly Pro Leu Ser His Ala Lys Asp Leu Trp
545 550 555 560
Ile His Gly Gly Tyr Asn Asn Trp Lys Asp Gly Leu Ser Ile Val Glu
565 570 575
Lys Leu Val Lys Ser Glu Arg Ile Asp Gly Asp Trp Trp Tyr Thr Glu
580 585 590
Val Val Ile Pro Asp Arg Ala Leu Val Leu Asp Trp Val Phe Ala Asp
595 600 605
Gly Pro Pro Lys His Ala Ile Ala Tyr Asp Asn Asn His Arg Gln Asp
610 615 620
Phe His Ala Ile Val Pro Lys His Ile Pro Glu Glu Leu Tyr Trp Val
625 630 635 640
Glu Glu Glu Leu Gln Ile Phe Lys Ala Leu Gln Glu Glu Arg Arg Leu
645 650 655
Arg Glu Glu Ala Met Arg Ala Lys Ala Glu Lys Ala Ala Arg Met Lys
660 665 670
Ala Glu Thr Lys Glu Arg Thr Met Lys Ser Phe Leu Leu Ser Gln Lys
675 680 685
His Ile Val Tyr Thr Glu Pro Leu Asp Val Gln Ala Gly Ser Ser Val
690 695 700
Thr Val Tyr Tyr Asn Pro Ala Asn Thr Val Leu Asn Gly Lys Pro Glu
705 710 715 720
Ile Trp Phe Arg Cys Ser Phe Asn Arg Trp Thr His Arg Leu Gly Pro
725 730 735
Leu Pro Pro Gln Lys Met Leu Pro Thr Glu Asn Gly Thr His Val Lys
740 745 750
Ala Thr Val Lys Val Pro Leu Asp Ala His Met Met Asp Phe Val Phe
755 760 765
Ser Glu Arg Glu Asp Gly Gly Ile Phe Asp Asn Arg Ser Gly Met Asp
770 775 780
Tyr His Ile Pro Val Phe Gly Gly Val Ala Lys Glu Pro Pro Met His
785 790 795 800
Ile Val His Ile Ala Val Glu Met Ala Pro Ile Ala Lys Val Gly Gly
805 810 815
Leu Gly Asp Val Val Thr Ser Leu Ser Arg Ala Val Gln Asp Leu Asn
820 825 830
His Asn Val Asp Ile Ile Leu Pro Lys Tyr Asp Cys Leu Lys Met Asn
835 840 845
Gln Val Lys Asp Phe Gln Phe His Lys Ser Tyr Phe Trp Gly Gly Thr
850 855 860
Glu Ile Lys Val Trp Phe Gly Lys Val Glu Gly Val Ser Val Tyr Phe
865 870 875 880
Leu Glu Pro Gln Asn Gly Leu Phe Trp Lys Gly Cys Val Tyr Gly Cys
885 890 895
Asn Asn Asp Gly Glu Arg Phe Gly Phe Phe Cys His Ala Ala Leu Glu
900 905 910
Phe Leu Leu Gln Gly Gly Phe His Pro Asp Ile Ile His Cys His Asp
915 920 925
Trp Ser Ser Ala Pro Val Ala Trp Leu Phe Lys Glu Gln Tyr Thr His
930 935 940
Tyr Gly Leu Ser Lys Ser Arg Ile Val Phe Thr Ile His Asn Leu Glu
945 950 955 960
Phe Gly Ala Asp Leu Ile Gly Lys Ala Met Thr Tyr Ala Asp Lys Ala
965 970 975
Thr Thr Val Ser Pro Thr Tyr Ser Arg Glu Val Ser Gly Asn Pro Val
980 985 990
Ile Ala Pro His Leu Tyr Lys Phe His Gly Ile Val Asn Gly Ile Asp
995 1000 1005
Pro Asp Ile Trp Asp Pro Leu Asn Asp Lys Phe Ile Pro Ile Ser Tyr
1010 1015 1020
Thr Ser Glu Asn Val Val Glu Gly Lys Thr Ala Ala Lys Glu Ala Leu
1025 1030 1035 1040
Gln Gln Lys Leu Gly Leu Lys Gln Ala Asp Leu Pro Leu Val Gly Ile
1045 1050 1055
Ile Thr Arg Leu Thr His Gln Lys Gly Ile His Leu Ile Lys His Ala
1060 1065 1070
Ile Trp Arg Thr Leu Glu Arg Asn Gly Gln Val Val Leu Leu Gly Ser
1075 1080 1085
Ala Pro Asp Pro Arg Ile Gln Asn Asp Phe Val Asn Leu Ala Asn Gln
1090 1095 1100
Leu His Ser Thr Tyr Asn Asp Arg Ala Arg Leu Cys Leu Thr Tyr Asp
1105 1110 1115 1120
Glu Pro Leu Ser His Leu Ile Tyr Ala Gly Ala Asp Phe Ile Leu Val
1125 1130 1135
Pro Ser Ile Phe Glu Pro Cys Gly Leu Thr Gln Leu Thr Ala Met Arg
1140 1145 1150
Tyr Gly Ser Ile Pro Ile Val Arg Lys Thr Gly Gly Leu Tyr Asp Thr
1155 1160 1165
Val Phe Asp Val Asp His Asp Lys Glu Arg Ala Gln Gln Cys Gly Leu
1170 1175 1180
Glu Pro Asn Gly Phe Ser Phe Asp Gly Ala Asp Ala Ala Gly Val Asp
1185 1190 1195 1200
Tyr Ala Leu Asn Arg Gln Leu Ser Cys Ser Ser
1205 1210

Claims (7)

1. A tobacco starch synthase gene is characterized in that the base sequence is specifically shown in SEQ ID NO. 1.
2. The tobacco starch synthase gene according to claim 1, wherein the amino acid sequence of the tobacco starch synthase gene is set forth in SEQ ID No. 2.
3. The tobacco starch synthase gene according to claim 1 or 2, characterized in that the method for preparing the tobacco starch synthase gene by PCR amplification comprises the steps of:
(1) Extracting genome and reverse transcribing the genome into cDNA for standby;
(2) Designing a primer for PCR amplification, and carrying out PCR amplification, wherein the specific primer sequence is designed as follows:
NtGLGA-F：5’-TTGGAGTTTCTTCTGCAAGGTG-3’，
NtGLGA-R：5’-CAATTACAGGGTTTCCAGACAC-3’。
4. a tobacco starch synthase gene according to claim 3, wherein, in extracting the genome in step (1), the leaf of the tobacco variety safflower grass is used as a sample.
5. Use of a tobacco starch synthase gene according to any one of claims 1 to 4, characterized in that the protein expressed by the gene is related to the starch content in tobacco leaves, and that the amylopectin and total starch content in the leaves is significantly reduced after the expression of the protein is reduced.
6. The use of the tobacco starch synthase gene according to claim 5, wherein the starch content in the tobacco is regulated and controlled by adjusting the expression level of the tobacco starch synthase gene using gene silencing techniques, or gene overexpression methods.
7. The use of the tobacco starch synthase gene according to claim 6, wherein the tobacco is transformed by constructing a virus-induced silencing vector, an RNAi interference vector, an overexpression vector or a genome editing vector containing the tobacco starch synthase gene by a transgenic technique, a transient expression technique or a genome editing technique, and screening to obtain a new variety of tobacco with a changed starch content.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981728A (en) * 1997-11-12 1999-11-09 Iowa State University Research Foundation Dull1 coding for a novel starch synthase and uses thereof
CN107759676A (en) * 2017-11-27 2018-03-06 南京农业大学 A kind of plant amylose synthesis associated protein Du15 and its encoding gene and application
CN110938639A (en) * 2019-12-19 2020-03-31 中国烟草总公司郑州烟草研究院 Tobacco ATP synthase gamma chain NtATPG and application thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5981728A (en) * 1997-11-12 1999-11-09 Iowa State University Research Foundation Dull1 coding for a novel starch synthase and uses thereof
CN107759676A (en) * 2017-11-27 2018-03-06 南京农业大学 A kind of plant amylose synthesis associated protein Du15 and its encoding gene and application
CN110938639A (en) * 2019-12-19 2020-03-31 中国烟草总公司郑州烟草研究院 Tobacco ATP synthase gamma chain NtATPG and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PREDICTED: Nicotiana tabacum soluble starch synthase 3, chloroplastic/amyloplastic-like (LOC107770809), transcript variant X1, mRNA;Zhang, H.等;GENBANK;1-2 *

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