阅读说明：本技术 一种利用OsPHR3基因在拟南芥中超表达提高植物氮磷吸收利用的方法 (Method for improving plant nitrogen and phosphorus absorption and utilization by overexpression of OsPHR3 gene in Arabidopsis thaliana ) 是由 孙雅菲 薛永 宋科 秦秦 孙丽娟 于 2020-05-19 设计创作，主要内容包括：本发明适用于农作物培育技术领域,提供了一种利用OsPHR3基因在拟南芥中超表达提高植物氮磷吸收利用的方法,通过从水稻幼苗叶片中提取总RNA,以oligodT为引物逆转录的cDNA为模板,克隆OsPHR3的编码区序列,然后将测序后确认完全正确的编码区序列连入含有CaMv35S启动子的pCAMBIA1305-GUS-Plus表达载体上,利用液氮冻融法将载体转入农杆菌感受态中,随后利用农杆菌侵染花序的方法实现拟南芥的遗传转化,将水稻基因OsPHR3转入拟南芥中,获得35S::OsPHR3拟南芥超表达植株。此发明可改良植株株形,并提高其对氮磷的吸收与利用,为改善植物对氮与磷营养元素的吸收利用提供技术支持,有良好的应用价值。(The invention is applicable to the technical field of crop cultivation, and provides a method for improving plant nitrogen and phosphorus absorption and utilization by overexpression of an OsPHR3 gene in Arabidopsis, which comprises the steps of extracting total RNA from rice seedling leaves, cloning a coding region sequence of OsPHR3 by using cDNA reverse transcribed by using oligodT as a primer as a template, connecting the completely correct coding region sequence after sequencing into a pCAMBIA1305-GUS-Plus expression vector containing a CaMv35S promoter, transferring the vector into an agrobacterium competence by using a liquid nitrogen freeze-thaw method, realizing genetic transformation of Arabidopsis by using a method of infecting a inflorescence by using agrobacterium, transferring the rice gene OsPHR3 into an OsPHR3 Arabidopsis super-expression plant of 35S. The invention can improve the plant shape of the plant, improve the absorption and utilization of nitrogen and phosphorus, provide technical support for improving the absorption and utilization of nitrogen and phosphorus nutrient elements of the plant, and has good application value.)

1. A method for improving plant nitrogen and phosphorus absorption and utilization by overexpression of an OsPHR3 gene in Arabidopsis thaliana is characterized by comprising the following steps: the method comprises the following steps:

s1, extracting total RNA from rice seedling leaves;

s2, cloning a coding region sequence of OsPHR3 by taking cDNA reverse transcribed by using oligo as a primer as a template, and then connecting the completely correct coding region sequence after sequencing into a pCAMBIA1305-GUS-Plus expression vector containing a CaMv35S promoter;

s3, transferring the vector into an agrobacterium-mediated state by using a liquid nitrogen freeze-thawing method, and further realizing the genetic transformation of arabidopsis by using a method of infecting inflorescences with agrobacterium to obtain an OsPHR3 arabidopsis overexpression plant.

2. The method for improving plant nitrogen and phosphorus absorption and utilization by over-expression of the OsPHR3 gene in Arabidopsis as claimed in claim 1, wherein the method comprises the following steps: the leaf of the rice seedling is cultured at the temperature of 30 ℃.

3. The method for improving plant nitrogen and phosphorus absorption and utilization by over-expression of the OsPHR3 gene in Arabidopsis as claimed in claim 1, wherein the method comprises the following steps: in step S1, the leaf age of the rice seedling is 14 days when total RNA is extracted.

Technical Field

The invention belongs to the technical field of crop cultivation, and particularly relates to a method for improving plant nitrogen and phosphorus absorption and utilization by overexpression of an OsPHR3 gene in Arabidopsis.

Background

The cultivation of crops with high nitrogen and phosphorus efficiency is an important way for maintaining the sustainable development of agriculture. The rice OsPHR3 is a member of a transcription regulatory factor PHR1 family (OsPHR1-4) with the most important phosphorus starvation response, the technology obtains an Arabidopsis transgenic material introduced with an exogenous gene OsPHR3 by tightening a transformation system infecting Arabidopsis inflorescence, and the technology has important significance for researching the mechanism of efficiently taking nitrogen and phosphorus nutrients by crops and the application of the gene in various plant species

People still choose to apply a large amount of nitrogen fertilizer and phosphate fertilizer in the agricultural production activities of China to ensure the normal growth of crops. However, the application of a large amount of fertilizer inevitably causes resource waste and environmental pollution. The cultivation of plants and new crop varieties with high nitrogen and phosphorus efficiency becomes an important way for maintaining the sustainable development of agriculture. Due to the unique physiological and biochemical characteristics of arabidopsis thaliana, arabidopsis thaliana protrudes from other plants and becomes an important plant vector for researching plant gene functions and gene application practices. However, when the nitrogen and phosphorus nutrients of plants are high-efficient, the efficient utilization of only one element is often aimed at, and the exploration of the synergistic high-efficient utilization of nitrogen and phosphorus and the excavation of the synergistic high-efficient transgenic plants are still deficient.

Disclosure of Invention

The invention provides a method for improving plant nitrogen and phosphorus absorption and utilization by overexpression of an OsPHR3 gene in Arabidopsis, which aims to improve plant shape, improve absorption and utilization of nitrogen and phosphorus by the plant, provide technical support for improving absorption and utilization of nitrogen and phosphorus nutrient elements by the plant, and have good application value.

The invention is realized in such a way that a method for improving plant nitrogen and phosphorus absorption and utilization by overexpression of an OsPHR3 gene in arabidopsis thaliana comprises the following steps:

s1, extracting total RNA from rice seedling leaves;

s2, cloning a coding region sequence of OsPHR3 by taking cDNA reverse transcribed by using oligo as a primer as a template, and then connecting the completely correct coding region sequence after sequencing into a pCAMBIA1305-GUS-Plus expression vector containing a CaMv35S promoter;

s3, transferring the vector into an agrobacterium-mediated state by using a liquid nitrogen freeze-thawing method, and further realizing the genetic transformation of arabidopsis by using a method of infecting inflorescences with agrobacterium to obtain an OsPHR3 arabidopsis overexpression plant.

Preferably, the leaf of the young rice plant is cultured at a temperature of 30 ℃.

Preferably, in step S1, the leaf age of the rice seedling is 14 days when the total RNA is extracted.

Compared with the prior art, the invention has the beneficial effects that: the invention relates to a method for improving plant nitrogen and phosphorus absorption and utilization by overexpression of an OsPHR3 gene in arabidopsis thaliana, which comprises the steps of extracting total RNA from rice seedling leaves, cloning a coding region sequence of OsPHR3 by taking cDNA reverse transcribed by oligo as a template, connecting the completely correct coding region sequence after sequencing into a pCAMBIA1305-GUS-Plus expression vector containing a CaMv35S promoter, transferring the vector into an agrobacterium-sensitive state by a liquid nitrogen freeze-thaw method, further realizing genetic transformation of arabidopsis thaliana by using an agrobacterium-infected inflorescence method to obtain an OsPHR3 arabidopsis thaliana overexpression plant, transferring the rice gene OsPHR3 into arabidopsis thaliana by an agrobacterium tumefaciens-mediated inflorescence dip-staining method to obtain an OsPHR3 overexpression strain, improving the plant shape, improving the absorption and utilization of nitrogen and phosphorus, and providing technical support for improving the absorption and utilization of nitrogen and phosphorus nutrient elements by plants, has good application value.

Drawings

FIG. 1 is a schematic diagram of molecular identification of OsPHR3 overexpression Arabidopsis thaliana in example 1 of the present invention.

FIG. 2 is a schematic diagram of the construction of the OsPHR3 overexpression vector and the vector in example 1 of the present invention.

FIG. 3 is a leaf phenotype representation of Arabidopsis wild-type and OsPHR3 overexpression lines of Experimental example 1 of the present invention.

FIG. 4 is a schematic diagram showing leaf lengths of Arabidopsis wild type and OsPHR3 overexpression lines of Experimental example 1 of the present invention.

FIG. 5 is a schematic diagram showing root system morphology of wild type Arabidopsis and OsPHR3 overexpression lines according to Experimental example 1 of the present invention.

FIG. 6 is a schematic diagram showing the length of siliques of the Arabidopsis wild type and OsPHR3 overexpression lines of Experimental example 1 of the present invention.

FIG. 7 is a diagram showing the phosphorus concentrations at different sites of the Arabidopsis wild-type and OsPHR3 overexpression lines of Experimental example 2 of the present invention.

FIG. 8 shows different NO's of Arabidopsis thaliana wild-type and OsPHR3 overexpression lines of Experimental example 3 of the present invention^3-NO at different sites under the treatment conditions^3-Concentration schematic.

FIG. 9 is a diagram showing the total nitrogen content of Arabidopsis thaliana wild-type and OsPHR3 overexpression lines of Experimental example 3 of the present invention under different phosphorus conditions.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.