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CN108118062B - Application of nitrate transport gene OsNRT1.9a in rice breeding - Google Patents

Application of nitrate transport gene OsNRT1.9a in rice breeding Download PDF

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CN108118062B
CN108118062B CN201711378092.7A CN201711378092A CN108118062B CN 108118062 B CN108118062 B CN 108118062B CN 201711378092 A CN201711378092 A CN 201711378092A CN 108118062 B CN108118062 B CN 108118062B
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方中明
黄玮婷
汪杰
朱炜
吕凯
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Abstract

本发明公开了硝酸根运输基因OsNRT1.9a在水稻选育中的应用，属于植物基因工程领域。OsNRT1.9a基因编码蛋白质的氨基酸序列如SEQ ID NO.1所示，cDNA序列如SEQ ID NO.2所示。本发明通过构建水稻OsNRT1.9a基因超表达植株、OsNRT1.9a基因干扰植株，发现通过提高OsNRT1.9a基因表达，可以使正常的水稻分蘖数增加，单株灌浆粒数和单株产量增加，因此OsNRT1.9a基因可用于水稻选育中以提高水稻产量。OsNRT1.9a基因在阐述氮素影响植物生长及发育过程方面以及在水稻株型改良方面具有重要的应用价值。

Figure 201711378092

The invention discloses the application of nitrate transport gene OsNRT1.9a in rice breeding, and belongs to the field of plant genetic engineering. The amino acid sequence of the protein encoded by the OsNRT1.9a gene is shown in SEQ ID NO.1, and the cDNA sequence is shown in SEQ ID NO.2. By constructing rice OsNRT1.9a gene overexpression plants and OsNRT1.9a gene interference plants, the present invention finds that by increasing the OsNRT1.9a gene expression, the normal rice tiller number can be increased, the number of grain filling per plant and the yield per plant can be increased, therefore OsNRT1.9a gene can be used in rice breeding to improve rice yield. The OsNRT1.9a gene has important application value in describing the effect of nitrogen on plant growth and development and in improving rice plant type.

Figure 201711378092

Description

Application of nitrate transport gene OsNRT1.9a in rice breeding
Technical Field
The invention belongs to the field of plant genetic engineering, and particularly relates to application of a nitrate transport gene OsNRT1.9a in rice breeding.
Background
The planting area of Chinese rice accounts for 20 percent of the total planting area of world crops, but the application amount of nitrogen fertilizer accounts for 37 percent of the total application amount of the world; in 1995, the production amount and the using amount of the nitrogen fertilizer reach the first world, but the using efficiency of the nitrogen fertilizer is low, the application amount of the nitrogen fertilizer is increased by 20 times than 50 years ago, and according to the trend, the application amount is predicted to be turned by 3 times again by 2050. Excessive application of nitrogen fertilizer can cause ecological pollution problems such as water eutrophication [ xu guo, fang xiao rong, functional research of rice nitrate transporter genes OsNRT1.1a and OsNRT1.1b, Nanjing university of agriculture, 2011: 4-6]. More nitrogen nutrients are wasted through denitrification, water and soil loss, natural volatilization, microbial utilization and the like.
If the nitrogen absorption efficiency is improved by 1%, the cost is saved by billions of dollars each year. From the analysis of Chinese conditions, the potential of enlarging the planting area and improving the total yield is limited, and the only way is to produce more rice on the limited land, namely, the yield per unit area is improved. In the conventional farming in the past, the nitrogen utilization efficiency is improved by selecting crops with higher nitrogen utilization efficiency; however, compared to breeding at the molecular level, this process appears slow and inefficient [ flood, its root, genotypic variation and physiological mechanism studies for nitrogen utilization in rice, university of Yangzhou, 2008: 10-13]. To improve nitrogen utilization, breakthrough must be sought from the molecular absorption mechanism of nitrogen. The family of nitrate transport genes is divided into two classes of low-affinity nitrate transport genes and high-affinity nitrate transport genes [ Zhousheyi, influence of sugars and amino acids on rice inducible high-affinity nitrate transport systems. 15-16]. Nitrate nitrogen and ammonium nitrogen are absorbed and converted into amino acids by nitrogen assimilation, which is called nitrogen first absorption. The seed nutrients are increased by the transport of nitrogen, increasing the degree of fullness, known as the second type of absorption of nitrogen, i.e. the reuse of nitrogen [ Kant S, Bi Y, Steven J, et al. unrestance plant stress to nitrogen limitation for the improvement of crop nitro use efficiency. journal of experiment, 2011,62(4):1499-1509 ]. The yield can be increased by increasing the nitrogen absorption accumulation amount or the nitrogen transport amount. Therefore, in modern agricultural construction, the utilization efficiency of the nitrogen fertilizer of rice is improved by a molecular breeding means, the pollution of the nitrogen fertilizer can be reduced, and the yield can be increased.
The NRT1/PTR family (NRT1/PTR family, NPF) refers to proteins capable of mediating transmembrane transport of substances such as nitrate and small peptides of 2-3 amino acid residues [ Rentsch D, Schmidt S, Tegeder M. Transporter for uptake and allocation of organic nitro compounds in plants FEBS Let,2007,581: 2281-. Members of the NRT1/PTR family are involved in the accumulation of proteins during seed formation and the transport of small molecule polypeptide forms following protein degradation during germination [ Martre P, Porter J R, Jamieson P D, et al. At present, few reports about NPF family member researches exist, and the OsNRT1.9 gene related by the invention is a nitrate transport gene of a rice NPF gene family. The invention discovers that the OsNRT1.9 gene can form two splicing types after being transcribed, wherein the first splicing type OsNRT1.9a has extremely important effect on rice tillering and can be applied to plant type improvement so as to increase the yield of rice.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides application of a nitrate transport gene OsNRT1.9a, a member of a rice NPF gene family, in rice breeding.
The purpose of the invention is realized by the following technical scheme:
the invention takes the NPF gene family member nitrate transport gene OsNRT1.9a of rice as an object, and clones the cDNA sequence of OsNRT1.9a from rice Zhonghua 11. The OsNRT1.9a gene overexpression vector is constructed, and the overexpression vector is introduced into the normal japonica rice variety middle flower 11 by adopting an agrobacterium EHA105 mediated genetic transformation method to obtain an OsNRT1.9a gene overexpression plant, wherein the tillering number, the effective spike number, the grain filling number and the yield of the OsNRT1.9a gene overexpression plant are obviously improved compared with those of a control wild type middle flower 11. An OsNRT1.9a gene interference expression vector is constructed through an RNAi technology, the interference expression vector is introduced into the middle flower 11, an interference plant with the OsNRT1.9a gene expression quantity reduced is obtained, and the tiller number, the effective spike number, the grain filling number and the yield of the interference plant are obviously reduced compared with those of the middle flower 11. These results indicate that by increasing the expression of the OsNRT1.9a gene, the number of tillers of normal rice can be increased, thereby increasing the number of ears, the number of grouted seeds and the yield of rice.
Based on the functions of the OsNRT1.9a gene discovered by the invention, the OsNRT1.9a gene can be used for rice breeding. The rice breeding is to improve the tillering number of rice, thereby improving the spike number, the grain number of the grouted seeds and the rice yield. Specifically, the tillering number of the rice, the number of ears per plant and the number of grouted seeds can be increased by improving the expression of the OsNRT1.9a gene, so that the aim of improving the yield of the rice is fulfilled.
The OsNRT1.9a gene can also be used for improving the yield of other plants, such as increasing the branch number of the plants by transgenically expressing the OsNRT1.9a gene in the plants, thereby improving the yield of the plants. The plant is monocotyledon or dicotyledon; such as: wheat, tomato, turf grass or alfalfa and the like.
The amino acid sequence of OsNRT1.9a protein coded by the OsNRT1.9a gene is shown as SEQ ID NO. 1; the cDNA sequence of the OsNRT1.9a gene is preferably shown as SEQ ID NO. 2.
It is understood that amino acid sequences having equivalent functions can be obtained by those skilled in the art by variously substituting, adding and/or deleting one or several amino acids of the amino acid sequence shown in SEQ ID NO.1 without affecting the OsNRT1.9a white activity (i.e., without the active center of the protein). Therefore, the OsNRT1.9a protein also includes proteins with equivalent activity obtained by substituting, replacing and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1. Furthermore, it will be appreciated that, given the degeneracy of codons and the preference of codons for different species, one skilled in the art can use codons suitable for expression in a particular species as desired.
The invention has the advantages and effects that:
(1) the tillering capability of rice is enhanced after the OsNRT1.9a gene cloned by the invention is over-expressed, which shows that the OsNRT1.9a gene is obvious to improve the yield of rice, therefore, the plant yield can be improved by improving the expression of the OsNRT1.9a gene through a genetic engineering technology. This not only facilitates the cultivation of high-yielding rice under normal nitrogen application conditions, but also allows the variety improvement of plants through molecular breeding.
(2) The successful cloning of the OsNRT1.9a gene further proves the important function of the NPF family in the nitrogen absorption process, has important significance for explaining the biological function of the NPF family, and has great promotion effect on further understanding the nitrogen metabolic pathway of plants and improving the nitrogen absorption efficiency.
(3) Although some genes have been cloned to improve plant yield, the molecular mechanisms for plant yield increase are still unclear. The OsNRT1.9a gene cloned by the invention can improve the yield of rice and has great promotion effect on determining key factors for increasing the yield of plants.
Drawings
FIG. 1 is a whole plant phenotype graph of 3 lines of the flower 11 and OsNRT1.9a gene overexpression plants in the control.
FIG. 2 is a statistical histogram of tillering counts of 3 lines of flowers 11 and OsNRT1.9a gene overexpression plants in the control, and the data were subjected to variable analysis (ANOVA) using SPSS software, and differential significance analysis was performed at three levels of 0.05, 0.01 and 0.001 using Duncan's, as indicated by "+," and "+" respectively, compared to the control.
FIG. 3 is a whole plant phenotype graph of 3 lines of the control floral 11 and OsNRT1.9a gene interference plants.
FIG. 4 is a statistical histogram of tillering counts of 3 lines of control floral 11 and OsNRT1.9a gene-disrupted plants, and the data were analyzed for variation (ANOVA) using SPSS software, and for differential significance using Duncan's at three levels, 0.05, 0.01 and 0.001, as compared to control.
FIG. 5 is a graph showing the results of measurement of the expression level of OsNRT1.9a gene in flower 11, OsNRT1.9a gene overexpression plants in control, and in OsNRT1.9a gene interference plants in 3 lines, and the data were subjected to analysis of variance (ANOVA) using SPSS software, and differential significance analysis was performed at three levels of 0.05, 0.01 and 0.001 using Duncan's, and expressed as:, and ×, respectively, compared with control.
FIG. 6 is a phenotype diagram of each grain filling of 3 lines of the flower 11, the OsNRT1.9a gene overexpression plant and the OsNRT1.9a gene interference plant in the control.
FIG. 7 is a statistical chart of the number of grain filled in each of the floral 11, the 3 lines of the OsNRT1.9a gene overexpression plant and the 3 lines of the OsNRT1.9a gene interference plant in the control. Data were analyzed for variables (ANOVA) using SPSS software, and for differential significance using Duncan's at three levels, 0.05, 0.01, and 0.001, expressed as x, and x, respectively, compared to controls.
Figure 8 is a statistical plot of the per plant yield of control flowers 11, 3 lines of osnrt1.9a gene overexpressing plants and 3 lines of osnrt1.9a gene interfering plants, the data were analyzed for variables (ANOVA) using SPSS software, and differential significance was analyzed at three levels of 0.05, 0.01 and 0.001 using Duncan's, as denoted by x, x and x, respectively, compared to the control.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental procedures used are conventional and can be carried out according to recombinant techniques already described (see molecular cloning, A laboratory Manual, 2 nd edition, Cold spring harbor laboratory Press, Cold spring harbor, N.Y.); the materials, reagents and the like used are all commercially available.
Example 1 construction of OsNRT1.9a Gene overexpression plant
Extracting RNA of rice middle flower 11, reversely transcribing the RNA into cDNA, and performing primer pair:
F1：5'-AGATCTATGGCCGCCATAGAAGAGGAG-3'(Bgl II)，
R1：5'-CTTAAGTCATGAAGCTGTGTTCTCTCT-3'(Afl II)；
after the cDNA of the OsNRT1.9a gene is amplified through PCR, the cDNA is cut by Bgl II and Afl II and then is connected into a pCAMBIA-1301 vector (the pCAMBIA-1301 vector is purchased from Cambia company), and a super-expression vector OsNRT1.9a-p1301 of the OsNRT1.9a gene is constructed. The overexpression vector is introduced into the flower 11 of a normal rice variety by adopting an agrobacterium EHA105 mediated genetic transformation method.
Transplanting all the obtained transgenic seedlings into a basket with soil, watering and fertilizing at regular intervals, planting the seedlings in a field when the seedlings grow to be about 10cm in height, extracting genome DNA (deoxyribonucleic acid) and detecting transgenic plants through PCR (polymerase chain reaction), wherein a detection primer pair is as follows:
F2：5'-GATGTTGGCGACCTCGTATT-3'，
R2：5'-TCGTTATGTTTATCGGCACTTT-3'。
if 517bp fragments are amplified, the transgenic plants are positive plants. And (4) harvesting and planting a single positive plant until a homozygous transgenic plant is identified at the T2 generation, namely obtaining the OsNRT1.9a gene overexpression plant. The tillering number of the OsNRT1.9a gene overexpression plant is far more than that of a flower 11 plant in a control, and the difference is obvious, as shown in figures 1 and 2. The expression level of OsNRT1.9a gene of the super-expression plant is detected, and the expression of the OsNRT1.9a gene is improved compared with that of a control, as shown in figure 5. Statistics of seeds collected from single plants shows that the grain filling amount of each plant of the over-expression plant is increased and the yield of each plant is increased, as shown in fig. 6, 7 and 8.
EXAMPLE 2 acquisition of OsNRT1.9a Gene interference plant
Extracting RNA of rice middle flower 11, reversely transcribing the RNA into cDNA, and performing primer pair:
F3：5'-GGTACCAGCCAGCCCTGAAGCACAGCAC-3'(Kpn I)，
R3：5'-GGATCCTGCACCACTCCCAAGGGCAGCA-3'(BamH I)；
F4：5'-ACTAGTAGCCAGCCCTGAAGCACAGCAC-3'(Spe I)，
R4：5'-GAGCTCTGCACCACTCCCAAGGGCAGCA-3'(Sac I)；
after cDNA fragments of the OsNRT1.9a gene are amplified by respective PCR, the cDNA fragments are cut by corresponding restriction enzymes and then are connected into a pTCK303 vector to construct an interference expression vector OsNRT1.9a-pTCK303 of the OsNRT1.9a gene. The interference expression vector is introduced into the normal japonica rice variety middle flower 11 by adopting an agrobacterium EHA105 mediated genetic transformation method.
Transplanting all the obtained transgenic seedlings into a basket with soil, watering and fertilizing at regular intervals, planting the seedlings in a field when the seedlings grow to be about 10cm in height, extracting genome DNA (deoxyribonucleic acid) and detecting transgenic plants through PCR (polymerase chain reaction), wherein a detection primer pair is as follows:
F2：5'-GATGTTGGCGACCTCGTATT-3'，
R2：5'-TCGTTATGTTTATCGGCACTTT-3'。
if 517bp fragments are amplified, the transgenic plants are positive plants. And (4) harvesting and planting a single positive plant until a homozygous transgenic plant is identified at the T2 generation, namely obtaining an OsNRT1.9a gene interference plant. The tillering number of the OsNRT1.9a gene interference plant is far less than that of a flower 11 plant in a control, and the difference is obvious, as shown in figures 3 and 4. The expression level of OsNRT1.9a gene in the interfering plant is detected, and the expression of the OsNRT1.9a gene is reduced compared with that of a control, as shown in FIG. 5. Statistics of seeds collected by single plants shows that the number of filled seeds of each plant of the interfering plants is reduced, and the yield of each plant is reduced, as shown in fig. 6, 7 and 8.
The results show that the tillering number of the rice can be increased by improving the expression of the OsNRT1.9a gene, and then the ear number and the rice yield are improved.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Wuhan bioengineering college
Application of <120> nitrate transport gene OsNRT1.9a in rice breeding
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gccatgtttg tgtattcttc cacgaggcac agagagaaca cagcttcatg a 1731

Claims (4)

1.OsNRT1.9a基因在水稻选育中的应用，其特征在于：所述的水稻选育为提高水稻分蘖数；所述的OsNRT1.9a基因编码的OsNRT1.9a蛋白的氨基酸序列如SEQ ID NO.1所示；通过提高OsNRT1.9a基因的表达实现所述应用。1. the application of OsNRT1.9a gene in rice breeding, it is characterized in that: described rice breeding is to improve rice tiller number; the amino acid sequence of the OsNRT1.9a protein encoded by described OsNRT1.9a gene is as SEQ ID NO .1; the application is achieved by increasing the expression of the OsNRT1.9a gene. 2.OsNRT1.9a基因在提高水稻穗数中的应用，其特征在于：所述的OsNRT1.9a基因编码的OsNRT1.9a蛋白的氨基酸序列如SEQ ID NO.1所示；通过提高OsNRT1.9a基因的表达实现所述应用。2. the application of OsNRT1.9a gene in improving the number of ears of rice, it is characterized in that: the amino acid sequence of the OsNRT1.9a protein encoded by the OsNRT1.9a gene is as shown in SEQ ID NO.1; by improving the OsNRT1.9a gene The expression realizes the application. 3.OsNRT1.9a基因在提高水稻灌浆粒数中的应用，其特征在于：所述的OsNRT1.9a基因编码的OsNRT1.9a蛋白的氨基酸序列如SEQ ID NO.1所示；通过提高OsNRT1.9a基因的表达实现所述应用。3. the application of OsNRT1.9a gene in improving grain number of rice grains, it is characterized in that: the amino acid sequence of the OsNRT1.9a protein encoded by the OsNRT1.9a gene is as shown in SEQ ID NO.1; by improving OsNRT1.9a The expression of the gene enables the application. 4.根据权利要求1-3任一项所述的应用，其特征在于：所述的OsNRT1.9a基因的cDNA序列如SEQ ID NO.2所示。4. The application according to any one of claims 1-3, wherein the cDNA sequence of the OsNRT1.9a gene is shown in SEQ ID NO.2.
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