Tobacco gene NtFIP1 for improving nitrogen utilization rate and cloning method and application of tobacco gene NtFIP1

A cloning method and a technology of utilization rate, applied in the application, genetic engineering, plant gene improvement and other directions, can solve the problem of not finding upstream regulatory genes, etc., and achieve the effect of improving the utilization rate of tobacco nitrogen and improving the quality of tobacco

Inactive Publication Date: 2019-11-12
JINING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, no upstream regulatory genes affecting the expression of tobacco nitrogen assimilation genes have been identified so far

Method used

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  • Tobacco gene NtFIP1 for improving nitrogen utilization rate and cloning method and application of tobacco gene NtFIP1
  • Tobacco gene NtFIP1 for improving nitrogen utilization rate and cloning method and application of tobacco gene NtFIP1
  • Tobacco gene NtFIP1 for improving nitrogen utilization rate and cloning method and application of tobacco gene NtFIP1

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Experimental program
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Effect test

Embodiment 1

[0046] The clone of the tobacco gene NtFIP1 coding sequence of the present invention was obtained according to the following method:

[0047] 1. Extraction of tobacco total RNA (kit method)

[0048] Use the kit method (column type total RNA extraction kit) to extract the total RNA of the corresponding tobacco plants, the specific method is as follows:

[0049] (1) Weigh about 100mg of tobacco variety K326 leaves in a 1.5mLEppendorf tube and grind them fully in liquid nitrogen;

[0050] (2) Add 1 mL of BufferRLT (the volume of the material is less than or equal to 10% of the volume of BufferRLT), shake and mix well, and let stand at room temperature for 5 minutes;

[0051] (3) To fully remove cell wall residues, proteins, fats, polysaccharides, etc., centrifuge at 4°C and 12000rpm for 10 minutes, and transfer the supernatant to a new centrifuge tube;

[0052] (4) Phase separation:

[0053] ①Add 0.2mL chloroform, shake vigorously for 15s, and let stand at room temperature for 2min;

[0054] ...

Embodiment 2

[0083] In this example, the methods for obtaining homozygous tobacco gene NtFIP1 complementary to Arabidopsis fip1 mutants and wild-type heterocomplementary lines and overexpression lines are as follows:

[0084] 1. Construction of pENTR3C-NtFIP1 recombinant plasmid

[0085] 1. Amplify the CDS of NtFIP1 gene by PCR and connect with pENTR3C

[0086] NtFIP1 forward primer (F): 5’-CGGGGTACCATGGAAGATGACGACG-3’;

[0087] NtFIP1 reverse primer (R): 5’-CCGCTCGAGATTGCTGGTCCATCTCCT-3’;

[0088] Using the extracted cDNA as a template, PCR amplification was carried out with specific primers F and R, and KpnI and XhoI restriction sites were introduced respectively upstream and downstream of the target gene. The PCR product was recovered with Tiangen (Beijing) glue recovery kit, and the PCR product was digested with Gateway-compatible pENTR3C. The operation steps are as shown in Table 4.

[0089] Table 4 The dosage of each ingredient during digestion

[0090] ingredient Dosage Target fragment r...

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Abstract

The invention belongs to the technical field of plant molecular biologies and gene cloning, and particularly relates to a tobacco gene NtFIP1 for improving the nitrogen utilization rate and a cloningmethod of the tobacco gene NtFIP1. The cloning of a coding sequence of the tobacco gene NtFIP1 is obtained, a recombinant expression vector containing the coding sequence of the tobacco gene NtFIP1 isprepared, the recombinant expression vector is transformed into an Arabidopsis fip1 mutant and a wild type, and heterogenous complementary transgenic plants and over expressed plants are obtained. Anexperiment shows that the tobacco gene NtFIP1 can be effectively expressed in the heterogenous complementary transgenic plants and the over expressed plants. The invention provides application of thetobacco gene NtFIP1. The tobacco gene NtFIP1 is applied to the plants to obtain transgenic plants to improve the nitrogen utilization rate of the plants, the NtFIP1 can be used for regulating the content of tobacco TSNAs, and thus the quality of tobacco is improved.

Description

Technical field [0001] The invention belongs to the technical field of plant molecular biology and gene cloning, and specifically relates to a tobacco gene NtFIP1 for improving nitrogen utilization efficiency and a cloning method and application thereof. Background technique [0002] Tobacco is an important economic crop in my country. Improving the quality of tobacco has become a strategic issue that my country’s tobacco industry urgently needs to solve. Tobacco is nitrate-nitrogen (NO 3 - ) Plants, due to the low nitrogen utilization rate in the production, and the different genotypes of tobacco for the nitrogen utilization rate are quite different, it is easy to cause the different types and contents of alkaloids in the tobacco to reduce the quality of tobacco leaves and affect the income of farmers. Therefore, research tobacco NO 3 - Metabolic processes such as absorption, transport, assimilation, and utilization to improve tobacco nitrogen utilization are the key to solving ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/10C12N15/82A01H5/00A01H6/20
CPCC07K14/415C12N15/10C12N15/8205C12N15/8243
Inventor 王超王大伟宋文路王勇杜昕昕权姝璇薛丽萍
Owner JINING UNIV
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