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Nitrate transport protein of diatom and its coding gene and application

A technology of nitrate transport and protein, applied in the field of nitrate transporter, can solve the problems of low utilization rate of nitrogen fertilizer, environmental pollution, etc., achieve the effect of improving nitrogen absorption capacity and broad application prospects

Inactive Publication Date: 2007-01-03
北京优利康生物农业技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In recent years, the amount of nitrogen fertilizer applied to various crops has been increasing, and the amount of application has seriously exceeded the standard. However, the utilization rate of nitrogen fertilizer in the soil by crops is very low, causing pollution to the environment and serious eutrophication of rivers.

Method used

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  • Nitrate transport protein of diatom and its coding gene and application
  • Nitrate transport protein of diatom and its coding gene and application
  • Nitrate transport protein of diatom and its coding gene and application

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Example 1. Cloning of diatom nitrate transporter gene NAT3

[0049] Use the following method to clone the nitrate transporter gene of diatoms, and the specific process includes the following steps:

[0050] 1. Extraction of total RNA from diatoms

[0051] Using TRIZOL from Invitrogen R Reagent kit (Cat.No.15596-026) and extracting the total RNA of diatoms with reference to the kit instructions, the specific method includes the following steps:

[0052] 1) Take 50-100mg of diatoms, grind them into powder in liquid nitrogen, and transfer them to a 1.5mL centrifuge tube.

[0053] 2) Add 1mL TRIZOL Reagent, mix thoroughly, and place at room temperature for 5 minutes.

[0054] 3) Add 200 μl of chloroform, shake for 15 seconds, place at room temperature for 2-3 minutes, and centrifuge at 12,000 g for 10 minutes at 4° C.

[0055] 4) Take the supernatant, add 500 μl of isopropanol, place at room temperature for 10 minutes, centrifuge at 12,000 g at 4° C. for 5 minutes, and...

Embodiment 2

[0078] Embodiment 2. Construct the plant expression vector of NAT3 and diatom NAT1 gene

[0079] 1. Construction of intermediate vector pCV121

[0080] see Figure 6 To construct the intermediate vector pCV121, the specific process includes the following steps:

[0081] 1. Construction of intermediate vector pUC121

[0082] The carrier pBI121 (Beijing Baierdi Biotechnology Co., Ltd.) was double-digested with restriction enzymes Sac I and EcoR I, and the 260bp T-NOS terminator fragment was recovered, which was combined with the vector double-digested with the same enzyme pUC19 (purchased from Dalian Bao Biological Co., Ltd.) was ligated to obtain the intermediate vector pUC121. It was identified by double enzyme digestion with restriction enzymes SacI and EcoRI, and the results of enzyme digestion identification were as follows: image 3 As shown (lane 1 is Marker: λDNA / EcoR I+Hind III, lanes 2 and 3 are enzyme digestion products), a DNA fragment with a size of about 260bp ...

Embodiment 3

[0090] Nitrogen reduction sensitivity experiment of embodiment 3.NAT3 transgenic tobacco

[0091] The plant expression vectors pNAT121 and pNAT1121 constructed in Example 2 were respectively transformed into Agrobacterium tumefaciens LBA4404 by the freeze-thaw method, and then the Agrobacterium tumefaciens LBA4404 transformants integrated with pNAT121 were transformed into tobacco NC89 by the leaf disc method, and positive transgenic plants were screened. Wherein with primer P1 (upstream primer): 5'-ATGAGTGGAACTGATGTTGCA-3' and P2 (downstream primer): 5'-TCTTCTCGGTATCAGGTTGGG-3' the result of PCR identification of NAT3 transgenic tobacco is as follows Figure 9 As shown (lane 1 is Marker: λDNA / EcoR I+Hind III, and lane 2 is the PCR product), positive transgenic plants can obtain a 1449bp band through PCR amplification, and as a result, 10 NAT3 and NAT1 transgenic tobacco plants were obtained. The seeds of NAT3, NAT1 transgenic tobacco and NC89 non-transgenic tobacco (control, ...

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Abstract

The present invention is kind of nitrate transport protein of diatom and its coding gene and application in transgenic plant. The nitrate transport protein has one of the following amino acid residue sequence: 1. SEQ ID No. 1 in the sequence list; and 2. the amino acid residue sequence of SEQ ID No. 1 through substitution, deletion or addition of 1-10 amino acid residues and coding protein possessing nitrate transport function. The transgenic tobacco, rice or lawn grass with the gene of the present invention NAT3 can grow normally on the culture medium with nitrogen element content only 1 / 32 normal value, and this proves the capacity of NAT3 in raising the nitrogen absorption. The present invention may have wide application foreground in plant nitrogen fertilizer absorption and breeding low nitrogen resisting plant variety.

Description

technical field [0001] The invention relates to a nitrate transporter derived from diatoms, a coding gene of the diatom nitrate transporter, and an application thereof in cultivating transgenic plants. Background technique [0002] Nitrogen fertilizer is an essential fertilizer for the growth of various crops (especially rice, lawn grass, tobacco, etc.). Rice is an important food crop in my country, accounting for 44% of the total grain output (Ling Qihong et al., On the irreplaceable role of rice production in the sustainable development of economically developed areas in southern my country, "China Rice", 2004 supplement). Nitrogen fertilizer is the most important fertilizer necessary for the growth of rice. With the increase of nitrogen fertilizer application rate, rice yield, tiller number, and total leaf number all have an obvious increasing trend (Pan Zhigao et al., Effects of different nitrogen fertilizer application rates on yield in rice intensive cultivation A Pre...

Claims

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Application Information

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IPC IPC(8): C07K14/405C12N15/31C12N15/82
Inventor 刘昱辉贾士荣伍祥贵包骏
Owner 北京优利康生物农业技术有限公司
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