Cloning of tobacco root-specific promoter and application thereof to transgenic plant

A specific promoter and specific gene technology, applied in the field of plant genetic engineering, to achieve the effect of solving rhizome diseases and insect pests, improving safety, safe and effective rhizome diseases and insect pests

Inactive Publication Date: 2010-11-03
FUJIAN AGRI & FORESTRY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]Because tobacco is tetraploid and its genetic background is extremely complex, it is very difficult to breed good cultivars. However, after years of cultivation, the main cultivated varieties are easy to produce Rhizome pests and diseases

Method used

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  • Cloning of tobacco root-specific promoter and application thereof to transgenic plant
  • Cloning of tobacco root-specific promoter and application thereof to transgenic plant
  • Cloning of tobacco root-specific promoter and application thereof to transgenic plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] [Example 1] extraction of tobacco leaf root and leaf total RNA

[0033]Plant the K326 original seed in a plate, and when the tobacco plant grows to a long period of prosperity, take the leaves of the tobacco plant at the 2nd, 3rd, 5th, 6th, and 9th leaves and wash them. 2. Divide the leaves at the inverted 3rd leaf position into two equal parts; divide the leaves at the inverted 5th and 6th leaf positions into 4 equal parts, and divide the leaves at the inverted 9th leaf position into four equal parts in the middle of the leaves, and take one part for each and freeze it in liquid nitrogen Store in -80°C refrigerator. After the leaves were sampled, the roots were washed quickly, and the white roots were frozen in liquid nitrogen and stored in a -80°C refrigerator. Take 2.5g root (experimental group Tester) and leaves (driver group Driver) in a mortar and add liquid nitrogen to grind, take 0.1g and pour it into pre-cooled 0.5ml guanidine isothiocyanate denatured homogena...

Embodiment 2

[0034] [Example 2] Construction of tobacco root and leaf cDNA suppression subtractive hybridization library

[0035] Detect the RNA quality that embodiment 1 extracts, with reference to the polyATtract of Promega company The mRNAIsolationSystemsIII step is to isolate mRNA, synthesize cDNA, and synthesize the first strand. Take 4ul of mRNA and mix it with 1ulcDNASynthesisPrimer; after short centrifugation, incubate at 70°C for 2min and immediately place it on ice for 2min. After short centrifugation, add 2ul of 5XFirst-StrandBuffer, dNTPMix ( 10mMeach) 1ul, sterileH2O1ul, AMVReverseTranscriptase (20units / ul)1ul, DEPCH2O5ul, mix well and centrifuge briefly, react at 42°C for 1 hour, put it on ice to stop the reaction, synthesize the second strand, add 48.4ul of sterileH2O to the first strand EP tube , 5XSecond-StrandBuffer16.0ul, dNTPMix (10mM)1.6ul, 20XSecond-StrandEnzymeCocktail4.0ul, after brief centrifugation, react at 16°C for 2hr; add 2ul (6U) of T4 DNA polymerase, mi...

Embodiment 3

[0039] [Example 3] Cloning and sequence feature analysis of tobacco root-specific genes

[0040] Prepare the cDNA of tobacco roots, stems and leaves respectively, prepare probes by digoxin labeling, hybridize and screen the DNA matrix membrane Macroarray made of tobacco root-specific expression library, take fluorescent photos, and compare the photos after digoxin-labeled hybridization, Find out the spots with differences, re-culture the single clone and send it for sequencing. figure 2 shown. The results showed that 130 clones with obvious differences were selected and sent for sequencing. The sequencing results were compared on NCBI, and 71 fragments had high homology with the published sequences of dicotyledonous plants such as tobacco and tomato. Among them, The differential gene fragment OG-12, after 5' and 3' RACE, obtained a full-length cDNA with a length of 848bp and a complete reading frame, using FGENESH (http: / / linux1.softberry.com / berry.phtml) Prediction analysi...

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Abstract

The invention provides cloning of a tobacco root-specific promoter and application thereof to a transgenic plant. The tobacco root-specific promoter at least comprises a nucleotide sequence showed in SEQIDNo.1. The method for cloning the tobacco root-specific promoter comprises the following steps: building an SSH library; manufacturing a probe; screening a specific gene by hybridization in the library; identifying spatial expression of the specific gene by RT-PCR; and cloning a full-length gene sequence and the upstream specific promoter. In the invention, the tobacco root-specific promoter is applied to the transgenic plant. The NtR12 root-specific promoter of the invention can improve the safety of tobacco transgenosis and effectively solve rhizome pests and diseases during the process of planting tobacco.

Description

technical field [0001] The invention relates to a method for cloning a tobacco root-specific expression promoter by means of genomics and functional genomics, and belongs to the technical field of plant genetic engineering. Background technique [0002] For a long time, tobacco rhizome diseases have been the main diseases that affect the yield and quality of tobacco, such as tobacco bacterial wilt, black shank, etc. Tobacco bacterial wilt is a devastating disease, and the bacteria invade from the root. The disease is currently mainly in the southern smoking areas of my country For example, Fujian, Hunan and other provinces generally occur, and the incidence rate in some plots reaches more than 80%, and it often occurs together with tobacco black shank. In severe cases, the whole field of tobacco can die (Liu Yong et al. 2007). It is known so far that R. solanacearum is harmful to XX species of XX family, but there is no specific medicine for the prevention and treatment of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10C12N15/82A01H5/00
Inventor 庄伟建陈顺辉闫利明蔡铁城潘建箐兰岚陈华
Owner FUJIAN AGRI & FORESTRY UNIV
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