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NtDR1 gene related with drought tolerance of plants and application of NtDR1 gene

A gene and plant technology, applied in the field of patent application of NtDR1 gene and its application, can solve problems such as slow progress of fine varieties

Active Publication Date: 2017-09-19
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the traditional hybrid breeding method, limited by the lack of resistant parental resources, the process of obtaining excellent varieties is very slow

Method used

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  • NtDR1 gene related with drought tolerance of plants and application of NtDR1 gene
  • NtDR1 gene related with drought tolerance of plants and application of NtDR1 gene
  • NtDR1 gene related with drought tolerance of plants and application of NtDR1 gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] This embodiment mainly focuses on NtDR1 The process of gene acquisition is briefly introduced as follows.

[0043] (1) Extraction of total RNA

[0044] Under normal growth conditions, take the cultivated tobacco K326 grown for 4 weeks, grind the sample material into powder in liquid nitrogen, and take about 100 mg of the powder material and place it in 1.5 ml containing 1.0 ml TRIzol (Invitrogen) Store in a centrifuge tube at -80°C for later use;

[0045] The total RNA is extracted by TRIzol reagent extraction method, the specific process is:

[0046] Take out the samples stored at -80°C for later use, after thawing at room temperature, add 200 μL of chloroform, shake and mix;

[0047] After centrifugation, carefully remove the upper aqueous phase, transfer it to another centrifuge tube, add 500 μL isopropanol, precipitate and centrifuge to separate RNA;

[0048] After washing with 75% alcohol and drying at room temperature, add appropriate volume of RNase-free water to fully dis...

Embodiment 2

[0072] Obtained using Example 1 NtDR1 Gene, the inventors further constructed a recombinant expression vector pCOsAc- NtDR1 , The relevant process is briefly introduced as follows.

[0073] First of all, obtained in Example 1 NtDR1 Connect the gene to the pCOsAc plasmid digested with Pmac I. Refer to Clontech's in-fusion seamless ligation instructions. According to the kit requirements, establish a ligation 10μL reaction system as follows:

[0074] 5x in-fusion, 2 μl;

[0075] pCOsAc (the product recovered after Pmac I digestion), 4 μl;

[0076] NtDR1 , 4μL;

[0077] Place on ice at 50°C for 15 min for the next transformation.

[0078] The heat shock method is used to transform the above-mentioned ligation product into E. coli competent cells. The specific process is:

[0079] Under aseptic conditions, take 10 μL of the ligation product and add it to the competent cells, mix gently and then ice bath for 30 minutes;

[0080] Heat shock at 42°C for 90s, quickly transfer the centrifuge tube t...

Embodiment 3

[0085] Using the heat shock method, the pCOsAc- prepared in Example 2 NtDR1 The vector was transformed into Agrobacterium GV3101 (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the plasmid was extracted and identified by restriction enzyme digestion to determine the expression vector pCOsAc -NtDR1 Successfully transformed Agrobacterium.

[0086] Transform Arabidopsis:

[0087] The expression vector was integrated into the Arabidopsis (Col-0) genome by Agrobacterium dip inflorescence method, and the selection was carried out on MS medium containing hygromycin (50mg / L);

[0088] Seeds of T2 generation continue to be screened on MS containing hygromycin, and the strains with a separation ratio of 3:1 are selected to continue screening in T3 generation;

[0089] At T3 generation, if 100% of the seeds can grow on MS with hygromycin, the strain is considered to be a homozygous strain.

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Abstract

The invention belongs to the technical field of tobacco gene engineering and particularly relates to an NtDR1 gene related with drought tolerance of plants and an application of the NtDR1 gene. The NtDR1 gene comprises a 921bp basic group with the basic group sequence shown in SEQ ID NO.1, and a specific nucleic acid sequence comprises 1st-921th nucleotides. The gene is expressed under drought induction, plays an important role in the drought tolerance process of tobacco and is highly related with drought tolerance of plants. Further, over-expression plasmid pCOsAc-NtDR1 is constructed and used for modifying plants, and an over-expression NtDR1 gene genetically modified gene plant is obtained. The over-expression NtDR1 gene genetically modified gene plant can remarkably increase the germination rate of modified plant seeds under the drought condition in the germination period, and in the seedling period and nutrition growth period, the genetically modified gene plant has more advantages in root length and root weight and shows better drought tolerance.

Description

Technical field [0001] The invention belongs to the technical field of tobacco genetic engineering, and specifically relates to a patent application for the NtDR1 gene related to plant drought tolerance and its application. Background technique [0002] Tobacco is a dicotyledonous plant of the genus Nicotiana in the family Solanaceae of the order Pipeflower. Tobacco has higher water requirements during the growth period, and if it suffers from drought stress, it will severely respond to tobacco leaf yield and quality. my country is a country with a relatively lack of water resources. Water resources are not evenly distributed in time and space. In recent years, some of our tobacco areas have frequently suffered severe droughts. The frequency and scope of the droughts have continued to expand, and the duration and losses suffered have increased. Had a great impact. [0003] Irrigation of water conservancy facilities is used to solve the problem of tobacco drought during the growing...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00
CPCC07K14/415C12N15/8273
Inventor 徐国云张慧翟妞陈千思刘萍萍申晓晔周会娜郑庆霞金立锋陈霞
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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