CN109207486B - Potato CISR2 gene and application thereof in low-temperature saccharification resistance - Google Patents
Potato CISR2 gene and application thereof in low-temperature saccharification resistance Download PDFInfo
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Abstract
The invention provides a potato CISR2 gene and application thereof in low temperature resistant saccharification; through a sequence published in a potato genome database, tuber cDNA of low-temperature saccharification resistant genotype potatoes stored for 15 days at 4 ℃ of 10908-06 is taken as a template, the CISR2 gene is cloned, an over-expression vector is constructed, and transgenic function verification is carried out by genetically transforming the potato No. 3, so that the capability of the over-expression CISR2 gene in resisting low-temperature saccharification can be improved.
Description
Technical Field
The invention belongs to the field of molecular genetic breeding, and particularly relates to a potato CISR2 gene and application thereof in low-temperature saccharification resistance.
Background
Potato (Solanum tuberosum L.) is a herbaceous plant of the genus Solanum of the family solanaceae, and is the fourth major food crop following wheat, corn and rice, and plays an important role in securing food safety and promoting economic development. At present, more than 150 countries around the world grow potatoes, second only to corn, and the second most widely distributed crop (xi shi hua, 2012). The consumption of edible potatoes in the world is changed from fresh potatoes to value-added potato processed foods, China is the first large production country of potatoes in the world, but special varieties for potato frying and the like are scarce, and the requirements of the development of the processing industry cannot be met (Jinliping and the like, 2003). In the potato processing industry, potato tubers are typically stored at temperatures below 10 ℃ in order to extend processing cycle time and prevent sprouting, desiccation shrinkage and disease transmission. However, low temperatures accelerate the conversion of starch to reducing sugars (glucose and fructose), i.e. low temperature saccharification. About 93% of the color variation of potato fried products is determined by the reducing sugar content (Roe et al, 1990; Pritcard and Adam, 1994). During the high temperature frying process, reducing sugars and free amino acids undergo Maillard Reaction, which darkens the fried products and produces acrylamide, a carcinogen harmful to the human body (Friedman, 2003; Tareke et al, 2002; Shepherd et al, 2010), a long-standing problem in the frying industry. It has been found that reducing acrylamide formation by reducing the level of reducing sugars in tubers is an effective approach (Amrein et al, 2003; Biedermann-Brem et al, 2003).
The problems existing in the prior art are as follows: the CISR2 transcription factor is screened out according to RNA-seq data and a co-expression experiment, and at present, no relevant literature is reported about the effect of CISR2 in the low-temperature saccharification and metabolism process of potato tubers.
Disclosure of Invention
In view of the defects of the prior art, the invention provides a potato CISR2 gene and application thereof in resisting low-temperature saccharification; through a sequence published in a potato genome database, a CISR2 gene is cloned by taking tuber cDNA of a low-temperature saccharification resistant genotype potato stored at 10908-.
In order to achieve the purpose, the invention adopts the following technical scheme:
potato RNA-seq analysis and low-temperature saccharification gene screening:
the invention selects a low-temperature saccharification resistant genotype '10908-06' and a low-temperature saccharification sensitive genotype 'Ejiang potato No. 3 (E3)' to plant in a greenhouse. After potato tubers are harvested, the potato tubers are placed in the shade at room temperature for 10 days, a single tuber with the weight of more than 50g and without diseases, insect pests and mechanical damage is divided into two parts, one part is stored at 20 ℃ for 0d, 5d and 15d, and the other part is stored at 4 ℃ for 5d and 15d, and then samples are respectively taken. When sampling, tubers are peeled, cut into small pieces, quickly frozen in liquid nitrogen, and stored at-70 ℃. The RNA of the above-mentioned samples was extracted by using a total RNA extraction kit for polysaccharide and polyphenol plants (DP441) from Tiangen Biochemical technology (Beijing) Ltd, and then subjected to transcriptome sequencing using Huada genes (refer to CN201510010458.X, CN201010213759.X, and CN 201610639522.5). The expression profile data of the low-temperature response of the low-temperature saccharification resistance and sensitive genotype storage tubers are analyzed, the expression pattern of the CISR2 in the resistant material is inconsistent, and the results of the expression profile data are shown in the following table (the expression amount of the gene is FPKM).
The potato CISR2 gene is cloned by the following steps:
(1) primer design
Primers for amplifying the CDS sequence of the gene were designed using SnapGene2.3.2 software based on the sequence information of the potato genome (PGSC0003DMG 400002620).
(2) RNA extraction and reverse transcription
The RNA of tubers stored for 15 days at 10908-064 ℃ of low-temperature saccharification resistance genotype potatoes is extracted by referring to a total RNA extraction kit of polysaccharide polyphenol plants of Tiangen (DP441), and cDNA is obtained by reverse transcription according to a TOYOBO reverse transcription kit (Code No. FSQ-101).
(3) PCR reaction and cloning vector construction
According to the synthesized primer, the CISR2 gene is amplified by PCR by taking the cDNA as a template, the amplified fragment is recovered by glue, TOPO cloning is carried out, bacterial liquid detection and sequencing are carried out, and the sequence is named as TOPO-CISR2 after being correctly sequenced.
The application of the potato CISR2 gene in low temperature resistant saccharification, the functional verification method comprises the following steps:
(1) plant expression vector construction
The TOPO-CISR2 plasmid and the over-expression vector pJCV55 are subjected to LR reaction, then transformed into Escherichia coli, detected by PCR and sequenced. The target vector with correct sequencing is named pJCV55-CISR 2.
(2) Genetic transformation of potato
The pJCV55-CISR2 plant expression vector was transformed into Agrobacterium strain LBA4404 and genetically transformed according to the method of Sihuaijun et al (2003).
(3) Low temperature resistant saccharification function verification
The obtained transgenic line and a control E3 test-tube seedling are planted in a greenhouse, tubers with fresh weights of more than 50g are respectively stored at 4 ℃ for 30 days after potato blocks are harvested, then sampling is carried out to determine the relative expression quantity, the reducing sugar content (RS content), the sucrose content (Suc content) and the acid invertase activity (VI activity) of CISR2, and the determination adopts a method of Zhang 2007.
Drawings
FIG. 1 shows the relative expression of CISR2 gene in transgenic tubers stored at 4 ℃ for 30 d;
FIG. 2 shows the reducing sugar and sucrose contents in transgenic tubers stored at 4 ℃ for 30 d;
FIG. 3 shows invertase activity in transgenic tubers stored at 4 ℃ for 30 d.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention are described in detail below with reference to the accompanying drawings. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the invention shown in the drawings and described in accordance with the drawings are exemplary only, and the invention is not limited to these embodiments.
It should be noted that, in order to avoid obscuring the technical solution of the present invention with unnecessary details, only the structures and/or processing steps closely related to the solution according to the present invention are shown in the drawings, and other details that are not relevant are omitted.
Example 1
The embodiment provides a potato CISR2 gene and a cloning method thereof, which specifically comprise the following steps:
(1) primer design
The CISR2 gene of the invention designs a primer to amplify the CDS full-length sequence of the gene according to the sequence (PGSC0003DMG400002620) published by a potato genome database, and the primer sequence is as follows:
CISR1F(5’-CCACATGGCGGAAGTTGAAGTACA-3’)
CISR1R(5’-TCAACAAGCCCATTTCAGTG-3’)
in addition, a quantitative primer of CISR2 is designed, and the primer sequence is as follows:
qCISR1F(5’-CCGCTAGGGCACATGATGTT-3’)
q CISR1R(5’-TTTGACAATGGACGAGGCAA-3’)
(2) RNA extraction and reverse transcription
The total RNA extraction kit of polysaccharide polyphenol plant of Tiangen (DP441) is used for extracting RNA of tubers of 10908-06 potatoes stored for 15 days at 4 ℃, and cDNA is obtained by reverse transcription according to TOYOBO reverse transcription kit (Code No. FSQ-101).
(3) PCR reaction and cloning vector construction
By a PCR method, a CDS sequence (741bp) of CISR2 is amplified by taking low-temperature saccharification resistant genotype potato 10908-06 as a template, and the reaction program is as follows: at 95 ℃ for 30 s; 95 ℃ for 15 s; 15s at 55 ℃; 72 ℃ for 30 s; 33 cycles (2-4 steps); 72 ℃ for 10 min; 4 ℃ forever. And recovering a corresponding target fragment from the gel, connecting the gel to a TOPO vector, then transforming the gel to escherichia coli DH5 alpha, carrying out spot picking and bacterial liquid detection, sending the gel to Weijie Jie (Shanghai) trade Limited company for sequencing verification, and extracting positive plasmids.
Example 2
In the embodiment, the CISR2 gene obtained in the embodiment 1 is subjected to low-temperature saccharification resistance verification, and the specific verification steps are as follows:
(1) plant expression vector construction
The TOPO-CISR2 plasmid and the over-expression vector pJCV55 are subjected to LR reaction, then transformed into Escherichia coli, detected by PCR and sequenced. The target vector with correct sequencing is named as pJCV55-CISR2, then the plasmid is transformed into agrobacterium LBA4404 through heat shock, and positive clones are obtained through bacterial detection.
(2) Genetic transformation of potato
The genetic transformation method of potato is carried out according to the method of Si et al (Si et al, 2003), the adopted agrobacterium strain is LBA4404, the acceptor material is potato E3, and the specific method comprises the following steps: 1) propagating the test-tube plantlet E3 of potato in an 8% MS solid culture medium (containing activated carbon), culturing in a potato growing room (22 ℃, 8h light/16 h dark light period), and using the test-tube potato growing for 6-8 weeks for genetic transformation; 2) marking agrobacterium containing target plasmid on LB solid culture medium (containing corresponding antibiotic) for activation, picking single clone in 20mLYEB liquid culture medium (containing corresponding antibiotic), culturing at 28 ℃ and 200rpm for 24h, taking 2mL bacterial liquid in 40mL YEB liquid culture medium (containing corresponding antibiotic), sub-culturing at 28 ℃ and 200rpm until OD600About 0.5 (about 6h), 5000rpm, centrifugation for 6min, supernatant removal, 10mL of 3% MS liquid medium heavy suspension; 3) adding a small amount of 3% MS liquid culture medium into a culture dish, and transversely cutting the test-tube potato in the dish to obtain potato tubes with the thickness of about 1-2 mm; putting the cut potato chips into the heavy suspension for infecting for 10min, slightly shaking midway, discarding bacteria liquid, putting the potato chips into a culture dish with filter paper, transferring the potato chips into a P1 co-culture medium (3% MS solid medium, 0.2mg/L GA3, 0.2mg/L IAA, 0.5 mg/L6-BA, 2mg/L ZT, pH 5.8) after the bacteria liquid is sucked to be dry, and culturing for 48h in a dark environment in a culture box at 23 ℃; 4) transferring the potato blocks subjected to dark culture to a P2 differentiation culture medium (3% MS solid culture medium +0.2mg/L IAA +0.2mg/L GA3+2mg/L ZT +0.5 mg/L6-BA +75mg/L Kan +400mg/L Cef), and culturing in an incubator at 23 ℃ (16h light/8 h dark light cycle); 5) the lateral buds of the potato pieces were subtracted after about 1 week. Resistant bud of potato pieceInoculated to a P3 rooting medium (3% MS +50mg/L Kan +400mg/L Cef), and after the resistant bud grows up, secondary rooting is carried out, and then positive detection is carried out.
(3) Low temperature resistant saccharification function verification
3 OE-CISR2(3, 5, 8) excess transgenic lines and a control E3 were planted in the greenhouse at the same time, and the transgenic lines and the control had no significant difference in field agronomic traits. The harvested transgenic tubers and the control E3 were stored in the dark at 20 ℃ for 10 days, and the tubers with a weight of 50g per plant were stored at 4 ℃ for 30 days, and then sampled.
Firstly, RNA of tubers stored at 4 ℃ for 30 days is extracted, cDNA is obtained through reverse transcription, the relative expression level of CISR2 is detected through real-time fluorescence quantitative RT-qPCR (see figure 1), and compared with a control, the expression level of CISR2 is obviously increased.
The content of reducing sugar is an important index for judging the low-temperature saccharification resistance of the potatoes, so that the content of the reducing sugar and the content of sucrose are measured for the treated transgenic strains and a reference, the content of the reducing sugar is measured by a dinitrosalicylic acid method, and the content of the sucrose is measured by the national ministry of health (GB5009, 8.85). After the OE-CISR2 hypertransgenic line tuber is stored for 30 days at 4 ℃, the content of reducing sugar is obviously reduced compared with the control (see figure 2), and the content of sucrose is not obviously different from the control (see figure 2). Meanwhile, the acid invertase activity of the treated transgenic lines and the control is determined, and the acid invertase activity is remarkably reduced when tubers of the OE-CISR2 hypertransgenic lines are stored at 4 ℃ for 30 days compared with the control (see figure 3). The existing experiment results show that the over-expression of the CISR2 gene greatly improves the low-temperature saccharification resistance of potatoes.
Compared with the prior art, the invention has the beneficial effects that:
(1) after the transgenic strains and tubers of the control E3 are stored for 30 days at 4 ℃, the reducing sugar content in OE-3, OE-5 and OE-8 strains is obviously lower than that in the control E3, which shows that the excessive expression of the CISR2 can effectively reduce the accumulation of reducing sugar under low-temperature storage.
(2) After the transgenic strains and tubers of the control E3 are stored for 30 days at 4 ℃, the activity of invertase in OE-3, OE-5 and OE-8 strains is obviously lower than that of the control E3, which indicates that the over-expression CISR2 gene can effectively inhibit the activity of acid invertase in the process of low-temperature storage.
The foregoing is directed to embodiments of the present application and it is noted that numerous modifications and adaptations may be made by those skilled in the art without departing from the principles of the present application and are intended to be within the scope of the present application.
Sequence listing
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<120> potato CISR2 gene and application thereof in low temperature saccharification resistance
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tcaaacaaat taccgaaagg gaagaaacgg ccgacgaaga aacagaaggc tattactaat 180
agtgagagta gacatcaaat ttacagagga gttaggatga gaagttgggg gaaatgggta 240
tctgaaattc gtgaaccgag gaagaaatca cgtatttggc tcggtactta tcctacagca 300
gaaatggccg ctagggcaca tgatgttgca gcggtgagta taaaaggaaa atccgccatt 360
ctcaatttcc ctcatctaat cgactcgttg cctcgtccat tgtcaaattc acctagagat 420
gttcaagctg ctgctgctaa agcagcatca atgagggacc caccttcttc agcctcatcg 480
tcctcgtcct cgtcctcatc ctcaacaaca tcaacgggat ctgaagagct ttgtgagatt 540
attgaactgc ctaatttagc agaaagtgac gattcaaaaa ctgagttgcg gccgagtgac 600
tcagttgaag gactgctgta ctcgccgtgg tgggcagatc atagtacaga tttttgtggt 660
tattttctgg agcagtctgc agctggcgct ggggaaagtt taatttcttg cagctttgag 720
acactgaaat gggcttgttg a 741
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Citations (5)
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|---|---|---|---|---|
| US5648249A (en) * | 1993-05-28 | 1997-07-15 | Monsato Company | Method of improving the quality of stored potatoes |
| CN102634497A (en) * | 2012-03-22 | 2012-08-15 | 华中农业大学 | Protein and gene for adjusting and controlling low temperature saccharification of potato as well as application thereof |
| CN104120131A (en) * | 2014-07-29 | 2014-10-29 | 华中农业大学 | Polynucleotide sequence for regulating low-temperature sweetening of potatoes and application thereof |
| CN105713991A (en) * | 2016-04-29 | 2016-06-29 | 华中农业大学 | Potato low-temperature sweetening resistant molecular marker combination and application thereof in potato low-temperature sweetening resistant breeding |
| CN107937591A (en) * | 2017-12-12 | 2018-04-20 | 华中农业大学 | The SNP marker of the Ⅺ relevant QTL site of end low-temperature resistance saccharification of tuberosum chromosomes and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| DE112010000876T5 (en) * | 2009-02-03 | 2012-09-13 | Wisconsin Alumni Research Foundation | Control cold-induced sweetening and reduce acrylamide levels in potatoes or sweet potatoes |
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| US5648249A (en) * | 1993-05-28 | 1997-07-15 | Monsato Company | Method of improving the quality of stored potatoes |
| CN102634497A (en) * | 2012-03-22 | 2012-08-15 | 华中农业大学 | Protein and gene for adjusting and controlling low temperature saccharification of potato as well as application thereof |
| CN104120131A (en) * | 2014-07-29 | 2014-10-29 | 华中农业大学 | Polynucleotide sequence for regulating low-temperature sweetening of potatoes and application thereof |
| CN105713991A (en) * | 2016-04-29 | 2016-06-29 | 华中农业大学 | Potato low-temperature sweetening resistant molecular marker combination and application thereof in potato low-temperature sweetening resistant breeding |
| CN107937591A (en) * | 2017-12-12 | 2018-04-20 | 华中农业大学 | The SNP marker of the Ⅺ relevant QTL site of end low-temperature resistance saccharification of tuberosum chromosomes and its application |
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| Distinct cold responsiveness of a StInvInh2 gene promoter in transgenic potato tubers with contrasting resistance to cold-induced sweetening;Xun Liu等;《Plant Physiol Biochem. 》;20161129;77-84 * |
| GenBank：XM_006342993;NCBI;《NCBI》;20160105;CDS部分和ORIGIN部分 * |
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