Slowly activating anion channel homologue NtSLAH5 for tobaccos and application of NtSLAH5

A channel protein and anion technology, applied in the field of tobacco slow anion channel protein NtSLAH5 and its application patent application, can solve the problems of large functional differences

Active Publication Date: 2019-03-26
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

but due to SLAC There are many genes in the gene family, and their functions vary greatly in different plants. Therefore, for different plants and different SLAC The function of the gene still needs to be further studied and differentiated, so that it can be targeted for future plant improvement

Method used

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  • Slowly activating anion channel homologue NtSLAH5 for tobaccos and application of NtSLAH5
  • Slowly activating anion channel homologue NtSLAH5 for tobaccos and application of NtSLAH5
  • Slowly activating anion channel homologue NtSLAH5 for tobaccos and application of NtSLAH5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] This example mainly focuses on the tobacco slow anion channel protein NtSLAH5 The gene acquisition process is briefly described as follows.

[0048] Taking cultivar tobacco leaves as samples, the total RNA of tobacco leaves was extracted by RNA extraction kit, and reverse transcribed into cDNA for future use;

[0049] The designed amplification primer sequences are as follows:

[0050] F: 5'-CGCGAGCTCGGTACCATGGTTAAAAGTGAATT-3',

[0051] R: 5'-GCTCACCATGGATCCTACTATTGATAACCG-3';

[0052] Using the above-prepared cDNA as a template, using the above-mentioned primers to carry out PCR amplification to obtain N tSLAH5 gene sequence.

[0053] The PCR reaction system is:

[0054] 1 μL of upstream primer,

[0055] Downstream primer 1 μL,

[0056] cDNA 1 μL,

[0057] 10×buffer 5μL,

[0058] dNTPs 6 μL,

[0059] EazyTaq enzyme 1 μL,

[0060] add ddH 2 0 to 50 μL.

[0061] PCR reaction program: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, anne...

Embodiment 2

[0070] for sure NtSLAH5 Gene function in tobacco, selection NtSLAH5 The specific nucleic acid fragment in the gene (the 1112-1448 nucleotide sequence of the sequence listing SEQID NO.1) was used as a guide sequence to construct a silent NtSLAH5 Transient silencing of genes used VIGS vector, and further transformed tobacco plants to construct transgenic plants. The relevant experimental procedures are briefly described as follows.

[0071] (1) Construction of VIGS vector for transient silencing

[0072] First, the primer sequences for PCR amplification were designed as follows:

[0073] NtSLAH5-F: 5'-TCGACGACAAGACCCTGCAGCAGTGGTGGGAAACTT-3',

[0074] NtSLAH5 -R: 5'-TGAGGAGAAGAGCCCTGCAGTGGAAAAGTGTAGGCCC-3';

[0075] Carry out PCR amplification with the above primer sequences (amplification length: 337bp) to obtain the guide sequence of VIGS;

[0076] Secondly, using the In-Fusion method, the above amplified guide sequence was ligated with the TRV vector (ligated at 50...

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Abstract

The invention belongs to the technical field of tobacco gene engineering and particularly relates to slowly activating anion channel homologue NtSLAH5 for tobaccos and patent application of the NtSLAH5. The CDS sequence of the gene contains an 1821bp basic group, the sequence of the basic group is represented by SEQ ID NO.1, and nucleotides 1112-1448 are specific nucleic acid fragments. The slowlyactivating anion channel homologue NtSLAH5 for the tobaccos is a key protein for the metabolism of chloride ions of the tobaccos, and the expression of the gene of the NtSLAH5 is inhibited by virtueof a virus induced gene silencing (VIGS) technique, so that the content of the chlorine ions in a transgenic silenced plant. Therefore, a good foundation can be laid for the cultivation of new speciesof low-chlorine-content tobaccos, and meanwhile, the basic technical support is provided for the quality stabilization of the tobacco leaves and the quality improvement of cigarettes.

Description

technical field [0001] The invention belongs to the technical field of tobacco genetic engineering, and in particular relates to a tobacco slow anion channel protein NtSLAH5 and a patent application for its application. Background technique [0002] Existing studies on tobacco generally believe that the appropriate chloride ion content in tobacco leaves is 0.3 to 0.8. When the content reaches 1%, it will affect the smoldering fire holding ability, and when it is further higher than 1%, the phenomenon of black ash flameout will occur. On the one hand, when the chloride ion content in tobacco leaves is too high, it will cause a lot of starch accumulation, the leaves are thick and brittle, and have high hygroscopicity, which makes the color easy to darken during storage and produces unpleasant odors. In conclusion, the chloride ion content in tobacco leaves has an important direct effect on the quality of tobacco leaves. [0003] Based on the direct influence and importance of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/10C12N15/82C07K14/415A01H5/00A01H6/82
CPCC12N15/8218C12N15/8243C07K14/415
Inventor 张慧徐国云卢鹏许亚龙翟妞金立锋周会娜李泽锋刘萍萍陈千思郑庆霞金静静
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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