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APDHI sequence of DNA unwindase gene of kender and its clone and applciation thereof

A technology of DNA helicase and apocynum, applied in the field of molecular biology and biology, can solve the problems of low salt tolerance function and limited improvement of salt tolerance

Inactive Publication Date: 2006-06-28
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the genetic engineering of salt-alkali-tolerant plants mainly focuses on these two aspects. Through transgenic technology, salt-alkali-tolerant transgenic plants have been obtained. However, the improvement of salt tolerance of these transgenic salt-tolerant plants is limited. The salt tolerance function of non-halophytes is lower than that of halophytes; on the other hand, the salt tolerance of plants is a biochemical metabolic process involving multiple genes, and the genes currently used only participate in one step of the biochemical metabolic process, so , Cloning genes involved in various metabolic pathways from halophytes, transforming non-halophytic crops, can further improve the salt tolerance of crops

Method used

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  • APDHI sequence of DNA unwindase gene of kender and its clone and applciation thereof
  • APDHI sequence of DNA unwindase gene of kender and its clone and applciation thereof
  • APDHI sequence of DNA unwindase gene of kender and its clone and applciation thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1: Cloning of Apocynum dna helicase gene APDH1

[0070] 1. The processing of Apocynum seedlings and the extraction of total RNA: the processing of Apocynum apocynum seedlings utilizing sand culture for 6 weeks was put into 800mM sodium chloride aqueous solution and distilled water for 2 days respectively, and RNA easy Plant mini Kit (Promoga company product) kit to extract total RNA from the leaves of Apocynum venetus seedlings treated with salt stress and treated with distilled water respectively.

[0071] 2. Construction and screening of suppression subtraction library: use Clontech SMART PCR cDNA synthesis kit to synthesize cDNA, treat seedling leaf cDNA with salt stress as tester, distilled water treated seedling leaf cDNA as driver, provide according to Clontech PCR-Select complementary DNA subtraction kit Apocynum salt stress inhibited subtractive cDNA library was constructed, and the positive cDNA clones screened from the subtractive library were diges...

Embodiment 2

[0074] Embodiment 2: the sequence of Apocynum DNA helicase gene APDH1 is as follows:

[0075] (1) Information of SEQ.ID.NO 1

[0076] (a) Sequential features

[0077] * Length: 1621 bp

[0078] *Type: nucleic acid

[0079] * Chain type: double chain

[0080] *Topology: Linear

[0081] (b) Molecular type: cDNA

[0082] (c) Assumption: No

[0083] (d) Antisense: no

[0084] (e) Original source: Apocynum

[0085] (f) Sequence description: SEQ.ID.NO 1

[0086] 1 attcttgtgc gaaaaccttt cttatccacc ttatctatat ctctccatcc tcaacattat

[0087] 61 agctcggtca acaagcatgg ccacaactac ttcggggccg gctaatcgta ggggaaccgt

[0088] 121 aatcgacgat aagctggtct ttgaaacgac cgaaggagtc gaggccatta cctccttcaa

[0089] 181 tggcatgggc ataaaagagg atttactccg tggtatctat gcttacggat tcgaaaagcc

[0090] 241 ttccgctata caacagcgag cggtaatgcc tatcatacag ggtcgagatg taattgcaca

[0091] 301 agctcaaagt ggtacgggta agaccagcat gattgccctt acagtatgcc aggtagttga

[0092] 361 tacttcggtg cgtgaagtcc aagcattaat ...

Embodiment 3

[0129] Embodiment 3: Construction and transformation of expression vector

[0130] 1. Gene isolation: according to the nucleotide sequence of the cloned Apocynum DNA helicase gene APDH1, design primers:

[0131] Forward primer: 5'-CCTTTCTTATCCACCTTATC-3'

[0132] Reverse primer: 5'-GCCAAAAACCCTAAACTATCAC-3'

[0133] The polymerase chain reaction was carried out using the cDNA reverse-transcribed from the total RNA of salt-stressed leaves as a template.

[0134] 2. Gene identification: Get 2 μl of PCR products and connect them with pGEM-T easy Vector. The method is carried out according to the instructions of Promega products. The connected products are transformed into DH5α bacteria (purchased from Shanghai Sangong Bioengineering Co., Ltd.), and the transformed bacteria contain ampicillin and / IPTG / -Gal LB solid medium culture, 37 ℃ inverted culture for 12-20 hours, some colonies are white, some turn blue. Select the white colony, extract the plasmid DNA, carry out enzyme d...

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Abstract

The invention relates to kendir DNA untwisting enzyme gene APDH1 clone, recombination, salt tolerance function analysis, and transgene salt and alkali proof cotton breeding selection. It belongs to molecular biology and biotechnology field. It includes the following steps; utilizing inhibition difference subtraction crossing technique to form kendir DNA positive direction difference subtraction cDNA library; processing enzyme cutting for positive cDNA clone, PCR and reverse direction Northern dot blot hybridization identification, DNA sequencing and nucleotide sequence homology comparison to gain cDNA sequence; one segment of the sequence is DNA untwisting enzyme gene middle segment; processing 3í»and 5í»end fast amplification to gain coding kendir DNA untwisting enzyme span cDNA named APDH1; and forming expression vector to transform cotton. The formed transgene cotton plant can normally come out, flower, and boll opening at 0.45% alkaline land.

Description

(1) Technical field [0001] The invention relates to the cloning, recombination, salt-tolerant function analysis of apocynum DNA helicase gene APDH1 and the breeding of transgenic salt-alkali-resistant cotton, which belongs to the field of molecular biology and biotechnology. (2) Background technology [0002] In my country's cultivated land, saline-alkali land is widely distributed. In addition, there are undeveloped saline-alkali wastelands. Due to poor drainage of irrigated land and seawater intrusion, the area of ​​secondary saline-alkali land in cultivated land is expanding year by year. Only various types of saline-alkali land in the Huanghuai-Hai Plain account for more than 50% of the cultivated land saline-alkali land. Soil improvement in saline-alkali land is costly and expensive. Breeding saline-alkali-tolerant crop varieties can not only economically and effectively increase crop yields in saline-alkali land and expand crop planting area, but also improve saline-a...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/14C12N15/82
Inventor 沈法富韩秀兰于元杰尹承佾
Owner SHANDONG AGRICULTURAL UNIVERSITY
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