Special carrier for improving utilization ratio of plant nitrogen nutrition and use thereof

A plant nitrogen, plant expression vector technology, applied in the field of special vector construction, can solve the problems of variable amino acid sequence and non-conservation, and achieve the effect of promoting growth and improving utilization efficiency

Inactive Publication Date: 2008-06-25
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The amino acid sequence of the C-terminal transcriptional regulatory domain is more variable and not conserved

Method used

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  • Special carrier for improving utilization ratio of plant nitrogen nutrition and use thereof
  • Special carrier for improving utilization ratio of plant nitrogen nutrition and use thereof
  • Special carrier for improving utilization ratio of plant nitrogen nutrition and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Embodiment 1, construction of intermediate vector pUC118-PrbcS-T

[0070] Purify (according to the instructions of the kit) pUC118-PrbcS-T-rbcS-3C with a plasmid extraction kit (Broad Tech), and pUC118-PrbcS-T-rbcS-3C (formed by Sugita et al. constructed and provided, rbcS-3C in Sugita et al., 1987, MGG, 209:247-256) was cut out, and the cut vector pUC118-PrbcS-T and rbcS-3C were separated by agarose gel electrophoresis Fragment, recover the 4.6kb vector pUC118-PrbcS-T, and then use the ligase kit of TaKaRa to ligate (operate according to the kit instructions) the vector DNA fragment without rbcS-3C to generate an intermediate vector pUC118-PrbcS -T (Figure 1), conversion of high efficiency (10 8 ) Escherichia coli competent cells (DH5α, Tiangen Biochemical Technology), spread the transformed Escherichia coli on a plate added with ampicillin (Amp, 100 μg / ml), cultivate overnight at 37°C, and screen Amp-resistant recombinants The plasmid was extracted from the Amp-resi...

Embodiment 2

[0071] Example 2, using point mutation technology to introduce NcoI site in the intermediate vector pUC118-PrbcS-T

[0072] Using the purified plasmid pUC118-Prbcs-T as a template, a pair of complementary primers (NcoI5 and NcoI3, Figure 1) for point mutations were designed according to the chloroplast localization sequence, and TaKaRa was commissioned to synthesize them. Add 25ng of purified plasmid pUC118-PrbcS-T to the point mutation reaction mixture as a template, and at the same time add 125ng of point mutation primers NcoI5 and NcoI3, 1μldNTP (2.5mM), 5μl of 10×KOD reaction buffer and 1μl of KOD polymerase (Toyobo Japan), add double distilled water to make the final reaction volume 50 μl. Heated at 95°C for 30 seconds on a PCR instrument, followed by 15 cycles of reaction at 95°C for 30 seconds, 55°C for 1 minute, 68°C for 10 minutes, and finally extended the reaction at 68°C for 10 minutes to synthesize The child chain of the mutation site. After the reaction is compl...

Embodiment 3

[0073] Example 3, construction of special vector pPZP221-PrbcS-Dof1

[0074] The construction strategy of the special vector pPZP221-PrbcS-Dof1 is shown in Figure 2. The first choice is to search for the full-length gene sequence of Arabidopsis Dof1 from GenBank, and design a pair of specific primers to amplify the Arabidopsis first-strand cDNA as a template. The full-length cDNA of Dof1 was obtained, the full-length gene fragment of Dof1 was recovered and purified, and connected to pMD18-T (Treasure Bioengineering Co., Ltd. (Dalian), TaKaRa) vector to obtain pMD-Dof1. Digest pMD-Dof1 and pUC118-PrbcS-*T with NcoI and XbaI, recover and purify the Dof1 gene fragment and the large vector fragment pUC118-PrbcS, and then connect and obtain the recombinant plasmid pUC118-PrbcS-Dof1. Digest pUC118-PrbcS-Dof1 and pPZP221 with HindIII and XbaI, recover and purify the small fragment PrbcS-Dof1 and the large fragment pPZP221, and then connect to obtain the recombinant vector pPZP221-Prb...

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Abstract

The invention discloses a special vector for improving utilization rate of plant nitrogen nutrition and application thereof. The vector of the invention comprises an arabidopsis transcription factor Dof1 gene, wherein, photoinduced promoters of Rubisco small subunits are arranged on upper reaches of the Dof1 gene. By using tobacco which is transformed from the special vector obtained by the invention to perform pot culture experiments under low, intermediate and high nitrogen nutritional conditions, results indicate that: height of a transgenic tobacco strain under the low nitrogen nutritional condition is 1.1 to 1.7 times of contrast tobacco; fresh weight of the transgenic tobacco strain is 1.05 to 1.3 times of the contrast tobacco; dry weight of the transgenic tobacco strain is 1.06 to 1.3 times of the contrast tobacco. Moreover, under the low nitrogen nutritional condition, activities of pyruvate kinase and phosphoenolpyruvate carboxylase of the transgenic tobacco are relatively 5 to 15 times of the contrast tobacco and 1.5 to 3.2 times of the contrast tobacco. Therefore, over expression of the Dof1 gene under the control of the photoinduced promoters can improve nitrogen utilization rate of the transgenic tobacco under the lower nitrogen nutritional condition and promotes growth of the transgetic tobacco. The special vector of the invention can improve nitrogen utilization efficiency of plants and has great application potential in genetic improvement for plant varieties.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to the construction and application of a special carrier for improving the utilization rate of nitrogen nutrition in plants. Background technique [0002] Nitrogen is an important nutrient element for plants. The forms of nitrogen absorbed by plants mainly include inorganic nitrogen (nitrate nitrogen and ammonium nitrogen) and organic nitrogen (such as urea, various amino acids, etc.). Although the absorption and utilization forms of various forms of nitrogen are different, after entering the plant, they must undergo transamination of glutamic acid or glutamine to form different amino acids, and finally synthesize proteins. Nitrogen absorption and assimilation are closely related to many physiological processes (such as photosynthesis, photorespiration, etc.), yield and quality of plants (Liu Wenguo, Fan Xue, Ma Anliang. 2002. Research progress on nitrogen absorp...

Claims

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Application Information

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IPC IPC(8): C12N15/82
Inventor 李昆志陈丽梅刘迪秋潘丽峰赵艳赵玥
Owner KUNMING UNIV OF SCI & TECH
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