Application of rice molybdate transporter coding gene OsMOT1 and 2

文档序号：163991 发布日期：2021-10-29 浏览：35次中文

阅读说明：本技术 水稻钼酸盐转运蛋白编码基因OsMOT1;2的应用 (Application of rice molybdate transporter coding gene OsMOT1 and 2 ) 是由黄新元胡大维赵方杰于 2021-08-24 设计创作，主要内容包括：本发明公开了水稻钼酸盐转运蛋白编码基因OsMOT1；2的应用。一种钼酸盐转运蛋白编码基因OsMOT1；2,序列如SEQ ID NO.1所示。该基因可在提高水稻地上部钼含量和钼由根系向地上部转运,提高水稻中钼由衰老组织向新生组织转移以及提高水稻籽粒钼含量等方面应用。本发明通过大量实验发现了水稻中的钼酸盐转运蛋白编码基因OsMOT1；2的生物学功能,该基因超表达后,水稻植株地上部钼含量显著上升,同时钼由根系向地上部转运比率显著提高；超表达植株中钼由衰老组织向新生组织转移增强；在水稻成熟阶段,超表达植株中籽粒钼含量、颖壳钼含量、枝梗钼含量、节间钼含量以及节钼含量显著提高。(The invention discloses a rice molybdate transporter coding gene OsMOT1; 2. A molybdate transporter encoding gene OsMOT1;2, the sequence is shown as SEQ ID NO. 1. The gene can be applied to the aspects of improving the molybdenum content of the overground part of the rice, transferring the molybdenum from the root system to the overground part, improving the molybdenum transfer in the rice from an aged tissue to a newborn tissue, improving the molybdenum content of rice grains and the like. According to the invention, a large number of experiments find that the molybdate transporter coding gene OsMOT1 in rice; 2, after the gene is over-expressed, the molybdenum content in the overground part of a rice plant is obviously increased, and meanwhile, the transport ratio of molybdenum from a root system to the overground part is obviously increased; the molybdenum in the over-expression plant is transferred and enhanced from the aged tissue to the newborn tissue; in the rice maturation stage, the molybdenum content of grains, the molybdenum content of glumes, the molybdenum content of branches, the molybdenum content of internodes and the molybdenum content of internodes in over-expression plants are obviously improved.)

1. Overexpression of a rice molybdate transporter coding gene OsMOT1;2, the application of improving the transport ratio of molybdenum from a rice root system to the overground part, and/or promoting the molybdenum to be transferred from a rice aging tissue to a new tissue, and/or improving the molybdenum content in a rice tissue organ, and is characterized in that the rice molybdate transporter coding gene OsMOT1;2 is shown in SEQ ID NO. 1.

2. The use of claim 1, wherein the rice tissue organ is one or more of grain, glume, branch, knot, internode.

3. The use according to claim 1, wherein the rice molybdate transporter coding gene OsMOT1;2 is shown in SEQ ID NO. 2.

4. An overexpression vector for improving the transport rate of molybdenum from a rice root system to the overground part, and/or promoting the molybdenum transfer from a rice aging tissue to a new tissue, and/or improving the molybdenum content in a rice tissue organ, wherein the overexpression vector contains the rice molybdate transporter coding gene OsMOT1 in claim 1; 2.

5. The overexpression vector according to claim 4, wherein the overexpression vector is a pUN1301-eGFP vector-based vector, and the molybdate transporter coding gene OsMOT1 shown in SEQ ID NO. 2; 2 is inserted between BamHI-SacI cleavage sites of the basic vector.

6. Use of the overexpression vector of claim 4 for increasing the transport rate of molybdenum from the root system of rice to the overground part, and/or promoting the transfer of molybdenum from the senescent tissue to the neogenetic tissue of rice, and/or increasing the molybdenum content in the tissue and organ of rice.

7. The use of claim 6, wherein the rice tissue organ is one or more of grain, glume, branch, knot, internode.

8. The use according to claim 6, wherein the overexpression vector of claim 4 is introduced into rice to obtain an overexpression molybdate transporter coding gene OsMOT1;2, thereby increasing the transport rate of molybdenum from the root system of the rice to the overground part, and/or promoting the molybdenum transfer from the aged tissue of the rice to the neogenetic tissue, and/or increasing the molybdenum content in the tissue and organ of the rice.

Technical Field

The invention belongs to the technical field of plant genetic engineering, and relates to a rice molybdate transporter coding gene OsMOT1; 2.

Background

Molybdenum (Mo) is one of the essential nutrients for the growth and development of almost all organisms, and is involved in many important physiological and biochemical metabolic processes in the life body. Plants lacking molybdenum cause leaf chlorosis, curling, blotches, reduced fruit set and poor seed development (Tejada-Jimeinenz et al, 2013), while human lacking molybdenum causes serious neurological disorders in young children, abnormal brain and head development and even death (Schwarz, 2005; Mendel & Kruse, 2012). Therefore, it is necessary to maintain the health of human body by enhancing the daily molybdenum intake of human body. Rice is one of the most important grain crops in the world, about half of the population in the world takes the rice as staple food, the absorption and transport mechanism of the rice to molybdenum is researched, the excellent rice variety with high molybdenum accumulation is cultivated, and the supplement of molybdenum nutrition from the dietary angle is an economic and effective solution.

At present, few reports of genes related to regulation and control of molybdenum transport in rice exist, people only have primary understanding on the process of molybdenum absorption from soil by rice roots, and the processes of wide transportation, reasonable distribution, efficient utilization and the like of molybdenum in rice plants are not clear. In particular, how is molybdenum absorbed into the rice body from the external environment promoted to be transported from the root system to the overground part, especially to the storage organ, i.e., the seed? How to promote the distribution of molybdenum nutrients among tissues and organs in the face of molybdenum deficiency, particularly the transfer from aged tissues to newborn tissues in rice? The existence of the problems limits people to improve the molybdenum content of rice grains by means of genetic engineering, so that the continuous excavation of the gene for regulating and controlling molybdenum transport in the rice has important practical significance.

Disclosure of Invention

The invention aims to solve the problems in the prior art and provides a rice molybdate transporter coding gene OsMOT1; 2. OsMOT1;2 accession number XM _015766127 in GenBank.

The first purpose of the invention is to provide a gene OsMOT1 for coding the overexpressed rice molybdate transporter; 2, improving the transport ratio of molybdenum from the rice root system to the overground part, and/or promoting the molybdenum to be transferred from the rice aged tissue to the new-born tissue, and/or improving the molybdenum content in the rice tissue organ, wherein the rice molybdate transporter coding gene OsMOT1;2 is shown in SEQ ID NO. 1.

Furthermore, the rice tissue organ is one or more of seeds, glumes, branches, knots and internodes.

Further, the rice molybdate transporter coding gene OsMOT1;2 is shown in SEQ ID NO. 2.

The second purpose of the invention is to provide an over-expression vector for improving the transport rate of molybdenum from the rice root system to the overground part, and/or promoting the molybdenum to be transferred from the rice aged tissue to the new tissue, and/or improving the molybdenum content in the rice tissue organ, wherein the over-expression vector contains the rice molybdate transporter coding gene OsMOT1; 2.

Further, the overexpression vector is a basic vector which is a pUN1301-eGFP vector, and a molybdate transporter coding gene OsMOT1 shown in SEQ ID No. 2; 2 is inserted between BamHI-SacI cleavage sites of the basic vector.

The third purpose of the invention is to provide the application of the overexpression vector in improving the transport rate of molybdenum from the root system of rice to the overground part, and/or promoting the molybdenum transfer from the aged tissues of rice to the newborn tissues, and/or improving the molybdenum content in the tissues and organs of rice.

Furthermore, the rice tissue organ is one or more of seeds, glumes, branches, knots and internodes.

Further, the application is that the overexpression vector is introduced into rice to obtain an overexpression molybdate transporter coding gene OsMOT1;2, thereby increasing the transport rate of molybdenum from the root system of the rice to the overground part, and/or promoting the molybdenum transfer from the aged tissue of the rice to the neogenetic tissue, and/or increasing the molybdenum content in the tissue and organ of the rice.

The invention has the advantages of

1. The invention provides a molybdate transporter coding gene OsMOT1 for the first time through systematic research; 2 biological function in molybdenum element transport.

2. The molybdate transporter coding gene OsMOT1;2 the content of molybdenum in yeast is obviously improved after the heterologous expression in the yeast, which indicates that the gene has molybdate transport activity.

3. The molybdate transporter coding gene OsMOT1;2, the molybdenum content of the overground part of the rice in the seedling stage is improved, the transport ratio of molybdenum from the root system to the overground part is improved, the molybdenum content of each leaf of the rice is improved, and meanwhile, the molybdenum content of the root system of the rice is not obviously influenced.

4. The molybdate transporter coding gene OsMOT1;2, in the environment of molybdenum deficiency, the molybdenum transfer from the aged tissue to the new tissue in the over-expression strain is obviously increased compared with the wild type, and the molybdenum content in the new tissue is obviously increased compared with the wild type.

5. The molybdate transporter coding gene OsMOT1;2, the molybdenum content, the glume molybdenum content, the branch molybdenum content, the internode molybdenum content and the molybdenum content of the rice seeds in the mature period are improved after overexpression.

6. A molybdate transporter coding gene OsMOT1 is constructed; 2.

The molybdate transporter coding gene OsMOT1 provided by the invention; 2, can effectively promote the molybdate to be transported to the overground part from the root system, particularly in the mature period of the rice, promote the molybdate to be transported to a storage organ, namely seeds, from the root system, can promote the distribution of molybdenum nutrition among tissues and organs, and particularly can promote the rice aging tissue to transfer to the new tissue when facing the environment lacking molybdenum.

Drawings

FIG. 1 shows the molybdate transporter encoding gene OsMOT1;2 in yeast, the content of molybdate in yeast cells is obviously increased after the heterologous expression in the yeast. EV means a yeast strain, OsMOT1, transformed into the vector pDR 196; 2, transferring into a recombinant vector pDR196-OsMOT 1;2, or a pharmaceutically acceptable salt thereof.

FIG. 2 shows the molybdate transporter encoding gene OsMOT1;2, the molybdenum content of the overground part of the rice in the seedling stage is obviously reduced after mutation, the molybdenum content of the root system is improved, and the transport ratio of molybdenum from the root system to the overground part and the molybdenum content of each blade of the rice are reduced.

A: in the nutrient solution containing 10nM molybdate, the molybdenum content of the overground part of the rice seedling growing to the four-leaf stage is obviously reduced compared with that of the wild type;

b: in the nutrient solution containing 10nM molybdate, the molybdenum content of the root system of the rice seedling growing to the four-leaf stage is obviously increased compared with that of the wild type;

c: in the nutrient solution containing 10nM molybdate, the transport ratio of molybdenum of the rice seedlings growing to the four-leaf stage from the root system to the overground part is obviously reduced compared with that of the wild type;

d: in the nutrient solution containing 10nM molybdate, the molybdenum content of each leaf of the rice seedling growing to the four-leaf stage is obviously reduced compared with that of the wild type.

FIG. 3 shows the molybdate transporter encoding gene OsMOT1;2, the molybdenum content of the overground part of the rice is improved, the transport ratio of molybdenum from the root system to the overground part is improved, the molybdenum content of each leaf of the rice is improved, and meanwhile, the molybdenum content of the root system of the rice is not obviously influenced.

A: wild type WT and OsMOT1;2 in the overexpression strain OsMOT1;2 relative expression level;

b: in a nutrient solution containing 10nM molybdate, the content of molybdenum in the overground part of the rice seedling growing to the four-leaf stage, OsMOT1;2 the content of molybdenum on the overground part of the over-expression strain is obviously increased compared with that of the wild type;

c: in a nutrient solution containing 10nM molybdate, the content of molybdenum in the root system of the rice seedling growing to the four-leaf stage, OsMOT1;2 the molybdenum content of the root system of the over-expression strain has no obvious difference with the wild type;

d: the transport ratio of molybdenum of rice seedlings growing to a four-leaf stage from root systems to overground parts in a nutrient solution containing 10nM molybdate, OsMOT1;2 the transport ratio of the overexpression strain is obviously increased compared with the wild type;

e: in a nutrient solution containing 10nM molybdate, the molybdenum content of each leaf of the rice seedling growing to the four-leaf stage, OsMOT1;2 the molybdenum content of each leaf of the over-expression strain is obviously increased compared with the wild type.

FIG. 4 shows the molybdate transporter encoding gene OsMOT1;2, in the environment of molybdenum deficiency, the molybdenum transfer from the aged tissue to the new tissue in the mutant is obviously reduced compared with the wild type, and the molybdenum content in the new tissue of the mutant is obviously reduced compared with the wild type.

A: growing the rice seedlings in a nutrient solution containing 10nM molybdate to a four-leaf stage, removing molybdenum nutrition in the nutrient solution, continuously culturing to a six-leaf stage, and comparing the molybdenum content of the same leaves of the rice in the four-leaf stage and the rice in the six-leaf stage, namely OsMOT1;2 the molybdenum transfer rate of each leaf in the mutant is lower than that of the wild type;

b: growing the rice seedlings in a nutrient solution containing 10nM molybdate to a four-leaf stage, removing the molybdenum nutrition in the nutrient solution, and continuously culturing to a six-leaf stage, namely OsMOT1; the molybdenum content of the sixth leaf of the newborn tissue in the 2 mutant is obviously lower than that of the wild type.

FIG. 5 shows the molybdate transporter encoding gene OsMOT1;2, in the environment of molybdenum deficiency, the molybdenum transfer of the over-expression strain from the aged tissue to the new tissue is obviously increased compared with the wild type, and the molybdenum content in the new tissue is obviously increased compared with the wild type.

b: growing the rice seedlings in a nutrient solution containing 10nM molybdate to a four-leaf stage, removing the molybdenum nutrition in the nutrient solution, and continuously culturing to a six-leaf stage, namely OsMOT1;2 the molybdenum content of the fifth leaf and the sixth leaf of the new tissue in the over-expression strain is obviously higher than that of the wild type.

FIG. 6 shows the molybdate transporter encoding gene OsMOT1;2, the molybdenum content of rice grains, the molybdenum content of glumes, the molybdenum content of branches and stems, the molybdenum content of joints, the molybdenum content of internodes, the molybdenum content of leaves and the molybdenum content of leaf sheaths are reduced after mutation.

A: OsMOT1 under potting conditions; 2, the molybdenum content and the glume molybdenum content of the mutant seeds are obviously lower than those of wild type seeds;

b: OsMOT1 under potting conditions; the molybdenum content of the branch and the stalk, the molybdenum content of the node, the molybdenum content of the internode, the molybdenum content of the leaf and the molybdenum content of the leaf sheath of the 2 mutant are all obviously lower than those of the wild type.

FIG. 7 shows the molybdate transporter encoding gene OsMOT1;2, the molybdenum content of rice grains, the molybdenum content of glumes, the molybdenum content of branches and stalks, the molybdenum content of knots and the molybdenum content of internodes are improved after overexpression.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit or essential characteristics thereof.

Example 1

A molybdate transporter encoding gene OsMOT1;2 heterologous expression in yeast, the specific implementation process is as follows:

1) amplifying OsMOT1 by using a PCR method by taking a cDNA library of a rice variety Zhonghua 11(ZH11) as a template; 2 (CDS sequence), OsMOT1;2 is shown as SEQ ID NO.2, and then the amplified OsMOT1;2 is connected between EcoRI-XhoI enzyme cutting sites of a pDR196 vector to construct a recombinant vector pDR196-OsMOT1 driven by a constitutive promoter PMA; 2.

the amplification primers are respectively:

OsMOT1；2-F:5'-CGGGCTGCAGGAATTCATGGCATCCTCCGCCGGCGAC-3′(SEQ ID NO.3)；

OsMOT1；2-R:5'-CGGGCCCCCCCTCGAGTCAAGCATCTCCAGCCCCAT-3′(SEQ ID NO.4)。

2) the recombinant vector and the empty vector were transformed into Saccharomyces cerevisiae 31019b (MATa ura3 mep 1. DELTA. mep 2. DELTA.: LEU2 mep 3. DELTA.: KanMX2), respectively. Selecting single clone to 2ml of molybdenum-free SD/-U broth and incubation overnight at 30 ℃ and 200rpm, then 1ml of the inoculum was transferred to 50ml of SD/-U broth without molybdenum and incubated at 30 ℃ and 200rpm up to the logarithmic growth phase. At this time, sodium molybdate was added to the cell suspension to make the molybdenum concentration in the cell suspension 0.5uM, the cell suspension was cultured for half an hour, and then the yeast cells were collected by centrifugation at 4000rpm at 4 ℃ using a centrifuge, and precooled EDTANa was used₂The solution is washed for three times, and finally, the solution is washed once by precooled ultrapure water.

3) Oven drying the fungus pieces, and weighing. After digestion of the samples in a graphite furnace, the molybdenum content of the 4 groups of samples was determined by ICP-MS.

The results show that the molybdate transporter coding gene OsMOT1;2 in yeast, the content of molybdate in yeast cells is obviously increased after the heterologous expression in the yeast. This example shows the molybdate transporter, OsMOT1;2 has molybdate transport activity (FIG. 1).

Example 2

A rice molybdate transporter coding gene OsMOT1;2, the specific implementation process of the mutant is as follows:

1) carrying out CRISPR/Cas9 gene editing technology on a molybdate transporter coding gene OsMOT1;2, knocking out. Selecting OsMOT1;2, a sequence CCGTCAAGTACATCCGCTAC (SEQ ID NO.5) and a sequence CGCTACTGTTCATAATCCTC (SEQ ID NO.6) of a CDS region are used as editing targets T1 and T2, and primers for constructing a knockout vector are designed.

The primer sequences are as follows:

gRT1:5’-TAGCGGATGTACTTGACGGGTTTTAGAGCTAGAAAT-3’(SEQ ID NO.7)；

OsU6aT1:5’-CCGTCAAGTACATCCGCTACGGCAGCCAAGCCAGCA-3’(SEQ ID NO.8)；

gRT2:5’-AGGATTATGAACAGTAGCGGTTTTAGAGCTAGAAAT-3’(SEQ ID NO.9)；

OsU6bT2:5’-CGCTACTGTTCATAATCCTCAACACAAGCGGCAGC-3’(SEQ ID NO.10)。

2) plasmid pYLsgRNA-OsU6a and plasmid pYLsgRNA-OsU6b were used as templates to amplify a sgRNA expression cassette fragment containing T1 and T2 by a PCR method.

The amplification system is as follows: 10 μ l Mix buffer; 2. mu.l of plasmid; guiding device1. mu.l of each substance; 6 μ l ddH₂O；

The amplification procedure was: (1) at 95 ℃ for 3 min; (2)95 ℃ for 15 s; (3) at 58 ℃ for 15 s; (4)72 ℃ for 20 s; (5)72 ℃ for 5 min; (6) 2min at 12 ℃; steps (2) - (4) were repeated for 35 cycles.

3) And taking the sgRNA expression cassette fragment in the step 2) as a template, and continuously amplifying complete sgRNA expression cassettes OsU6a-T1-sgRNA and OsU6 a-T2-sgRNA.

The amplification system is as follows: 10 μ l Mix buffer; 2. mu.l of the expression cassette fragment; 1. mu.l of each primer; 6 μ l ddH₂O；

4) sgRNA expression cassettes OsU6a-T1-sgRNA and OsU6a-T2-sgRNA were assembled into a pYLCRISPR/Cas9 knock-out vector.

5) The pYLCRISPR/Cas9 plasmid that confirmed the correctness was transformed into rice. The specific transgenic process is as follows: (1) the pYLCRISPR/Cas9 plasmid was transformed into Agrobacterium (GV 3101); (2) taking rice variety ZH11 seeds as materials, shelling, sterilizing, and placing on an induction culture medium to induce callus; (3) infecting the callus with agrobacterium liquid, washing with sterile water, and setting in selective culture medium to screen out resistant callus; (4) transferring the resistant callus to a differentiation medium and a rooting medium in sequence, and obtaining transgenic T0 generation plants after induced differentiation and rooting.

6) Planting T0 generation plants in a field, and identifying a molybdate transporter coding gene OsMOT1;2, selecting homozygous mutant individuals and harvesting seeds of T1 generations.

The identification primer is as follows:

CRISPROs1；2-F:5'-CCCCATGCCCGTCCAGCCCAT-3′(SEQ ID NO.11)；

CRISPROs1；2-R:5'-CGGCGCCCTCCCAGAACCCGATCT-3′(SEQ ID NO.12)；

through detection, three molybdate transporter coding genes OsMOT1 are obtained in the embodiment; 2, a knockout mutant osmot1 with a different editorial form; 2-1, osmot1; 2-2 and osmot1; 2-3.

Example 3

The molybdate transporter-encoding gene OsMOT1 prepared in example 2; 2 mutant osmot1; 2-1, osmot1; 2-2 and osmot1; 2-3, the concrete implementation process of the water culture experiment is as follows:

1) contacting wild-type WT with OsMOT1;2 seeds of the mutant were germinated at 37 ℃ for 2 days, sown on a plastic black net suspended in tap water, and after one week, the seedlings were transferred to 1/2Kimura B nutrient solution containing 10nM molybdate to continue culturing to the four-leaf stage of rice, during which the nutrient solution was changed every three days.

2) Rinsing wild type and mutant osmot1 with tap water; 2-1, osmot1; 2-2, osmot1; 2-3, removing the nutrient solution attached to the surface of the root system and the base part of the stem, then washing the plant for three times by using ultrapure water, and sampling according to different leaves and root systems.

3) And drying the sample and weighing. And (4) digesting the sample by using a graphite furnace, and measuring the molybdenum content of the sample by using ICP-MS.

This example shows the molybdate transporter encoding gene OsMOT1;2 after mutation, the molybdenum content in the overground part of the rice, the molybdenum content in each leaf and the molybdenum transport ratio from the root system to the overground part are all obviously reduced, while the molybdenum content in the root system is obviously increased, namely the molybdenum transport ratio from the root system to the overground part and the molybdenum content in each leaf of the rice are reduced (figure 2).

Example 4

A rice molybdate transporter coding gene OsMOT1;2, constructing a overexpression vector, and specifically implementing the following steps:

1) taking a ZH11 cDNA library as a template, and adopting pUN1 shown in SEQ ID NO.13 and SEQ ID NO. 14; 2-1 primer amplifying OsMOT1;2 (shown as SEQ ID NO. 2), and pUN1301-eGFP plasmid is used as a template, and pUN1 shown as SEQ ID NO.15 and SEQ ID NO.16 is adopted; 2-2 primers amplify eGFP sequences.

The amplification primers are as follows:

pUN1；2-1F：5'-CGACTCTAGAGGATCCATGGTGAGCAAGGGCGAG-3′(SEQ ID NO.13)；

pUN1；2-1R：5'-GATGCCATCTTGTACAGCTCGTCCATGCC-3′(SEQ ID NO.14)；

pUN1；2-2F：5'-GTACAAGATGGCATCCTCCGCCG-3′(SEQ ID NO.15)；

pUN1；2-2R：5'-GATCGGGGAAATTCGAGCTCTCAAGCATCTCCAGCCCCATC-3′(SEQ ID NO.16)；

the amplification system is as follows: 10 μ l Mix buffer; 2. mu.l of cDNA or plasmid; 1. mu.l of each primer; 6 μ l ddH₂O；

The amplification procedure was: (1) at 95 ℃ for 3 min; (2)95 ℃ for 15 s; (3) 15s at 55 ℃; (4)72 ℃ for 90 s; (5)72 ℃ for 5 min; (6) 2min at 12 ℃; steps (2) - (4) were repeated for 35 cycles.

2) And carrying out enzyme digestion on the plasmid pUN1301-eGFP to obtain a linearized plasmid.

The restriction enzyme sites are: BamHI-SacI;

the enzyme cutting system is as follows: 1 μ g of plasmid; 1. mu.l of each enzyme; 5 μ l 10 Xbuffer; supplemental ddH₂O to a total of 50. mu.l;

the enzyme digestion program is as follows: (1) 4h at 37 ℃; (2)4 ℃ for 2 min.

3) Connecting the amplified sequence to the BamHI-SacI enzyme cutting sites of the pUN1301-eGFP vector by a homologous recombination method, and constructing a recombinant vector pUN1301-eGFP-OsMOT1 driven by a constitutive promoter Ubiquitin; 2.

the recombinant is: 4. mu.l of linearized vector; 2. mu.l of each fragment; 4 μ l of 5 Xbuffer; 2. mu.l of enzyme; 6 μ l ddH₂O；

The recombination program is as follows: (1) 30min at 37 ℃; (2)4 ℃, 30 s;

through detection, the overexpressed rice molybdate transporter coding gene OsMOT1 is obtained in the embodiment; 2 recombinant vector pUN1301-eGFP-OsMOT 1; 2.

example 5

A molybdate transporter encoding gene OsMOT1;2, constructing the over-expression material, and specifically implementing the following steps:

1) the recombinant vector pUN1301-eGFP-OsMOT1 prepared in example 4; 2 into rice, the transgenic process was the same as in example 2. T0 generation plants are planted in the field, and T1 generation seeds are harvested.

2) Accelerating germination of T1 generation seeds at 37 ℃ for 2 days, sowing the seeds on a plastic black net suspended in tap water, transferring seedlings to 1/2Kimura B nutrient solution after one week for continuous culture, and carrying out positive identification on T1 generation plants by a GUS staining method.

3) Taking positive single plant and wild WT plant leaves, extracting RNA, and obtaining cDNA after reverse transcription.

4) Detecting a molybdate transporter coding gene OsMOT1 in wild type and positive individual plants by using fluorescent quantitative PCR; 2, calculating OsMOT1 by taking the rice housekeeping gene Actin as an internal reference; 2 relative expression level.

OsMOT1;2 is as follows:

qOsMOT1；2-F:5'-CTCATGAATTTCGTGGGGTG-3′(SEQ ID NO.17)

qOsMOT1；2-R:5'-AGCATGACGCCCAGTATC-3′(SEQ ID NO.18)

the quantitative primers of Actin are:

Act-rts:5'-TGGTCGTACCACAGGTATTGTGTT-3′(SEQ ID NO.19)

Act-rta:5'-AAGGTCGAGACGAAGGATAGCAT-3′(SEQ ID NO.20)。

through detection, two molybdate transporter coding genes OsMOT1 are obtained in the embodiment; 2 OsMOT1 with different relative expression amounts; 2 overexpression lines OE-1 and OE-2 (FIG. 3A).

Example 6

The molybdate transporter-encoding gene OsMOT1 prepared in example 5; 2 the water culture experiment of over-expression strains OE-1 and OE-2 is implemented in the following specific steps:

1) contacting wild-type WT with OsMOT1;2 seeds of the over-expression line were germinated for 2 days at 37 ℃ and sown on a plastic black net suspended in tap water, and after one week, the seedlings were transferred to 1/2Kimura B nutrient solution containing 10nM molybdate and cultured continuously until the four-leaf stage of the rice, during which the nutrient solution was changed every three days.

2) The wild type and mutant OE-1 and OE-2 plants were rinsed with tap water to remove the nutrient solution attached to the surface of the root and stem bases, then rinsed three times with ultrapure water, and sampled for different leaves and roots.

3) And drying the sample and weighing. And (4) digesting the sample by using a graphite furnace, and measuring the molybdenum content of the sample by using ICP-MS.

The results show that: this example shows the molybdate transporter-encoding gene OsMOT1 in OE-1 and OE-2; 2, the molybdenum content in the overground part of the rice, the molybdenum content in each leaf and the transport ratio of molybdenum from the root system to the overground part are all obviously increased, and the molybdenum content in the root system is not obviously changed (figures 3B-E).

Example 7

A molybdate transporter encoding gene OsMOT1;2, carrying out a water culture experiment under the condition of lacking molybdenum of the mutant, wherein the concrete implementation process is as follows:

1) preparing OsMOT1;2 the seeds of the mutant were manipulated as in example 2. Contacting wild-type WT with OsMOT1;2 seeds of the mutant were germinated at 37 ℃ for 2 days, sown on a plastic black net suspended in tap water, and after one week, the seedlings were transferred to 1/2Kimura B nutrient solution containing 10nM molybdate to continue culturing to the four-leaf stage of rice, during which the nutrient solution was changed every three days.

2) Contacting wild type with OsMOT1;2 mutant plants were removed and incubated with 1/2Kimura B nutrient solution without molybdenum to create a molybdenum deficient environment until the six-leaf stage of the rice, during which the nutrient solution was changed every three days.

3) Flushing wild type and mutant plants osmot1 with tap water; 2-1, osmot1; 2-2, osmot1; and 2-3, removing the nutrient solution attached to the surfaces of the root system and the stem base, then cleaning the root system and the root system for three times by using ultrapure water, and taking the aged and newly grown leaves and the root system as samples respectively.

4) And drying the sample and weighing. And (4) digesting the sample by using a graphite furnace, and measuring the molybdenum content of the sample by using ICP-MS.

This example shows the molybdate transporter encoding gene OsMOT1;2 mutation, in the molybdenum-deficient environment, the transfer rate of molybdenum from senescent tissue to neogenetic tissue in the mutant was significantly reduced compared to the wild type, while the molybdenum content in the neogenetic tissue of the mutant was significantly reduced compared to the wild type (fig. 4).

Example 8

The molybdate transporter-encoding gene OsMOT1 prepared in example 4; 2, carrying out a water culture experiment under the condition of lack of molybdenum of over-expression strains OE-1 and OE-2, wherein the specific implementation process is as follows:

2) Contacting wild type with OsMOT1;2 the plants of the over-expression line were removed and a molybdenum-deficient environment was created with 1/2Kimura B nutrient solution without molybdenum, and the culture was continued until the six-leaf stage of the rice, during which the nutrient solution was changed every three days.

3) The wild type and the super expression line plants OE-1 and OE-2 are washed by tap water to remove the nutrient solution attached to the surface of the root system and the base of the stem, and then washed by ultrapure water for three times, and the aged and newly grown leaves and root systems are taken as samples respectively.

4) And drying the sample and weighing. And (4) digesting the sample by using a graphite furnace, and measuring the molybdenum content of the sample by using ICP-MS.

This example shows the molybdate transporter encoding gene OsMOT1;2, in the molybdenum-deficient environment, the transfer rate of molybdenum from the senescent tissue to the neogenetic tissue in the overexpression strain is obviously improved compared with that of the wild type, and the molybdenum content in the neogenetic tissue of the overexpression strain is obviously improved compared with that of the wild type (figure 5).

Example 9

Molybdate transporter coding gene OsMOT1 under potting conditions; 2, the specific implementation process of the molybdenum content determination of the mutant is as follows:

1) preparing OsMOT1;2 the seeds of the mutant were manipulated as in example 2. Contacting wild-type WT with OsMOT1;2, accelerating germination of seeds of the mutant at 37 ℃ for 2 days, sowing the seeds on a plastic black net suspended in tap water, culturing for two weeks, transplanting seedlings into black plastic barrels filled with 5kg of soil, repeating the steps one by one in each barrel, totaling 6 barrels, placing the barrels in a greenhouse, and managing water and fertilizer according to normal field management;

2) harvesting wild type and OsMOT1 in the mature period of rice; 2 mutant osmot1; 2-1, osmot1; 2-2, osmot1; 2-3, sampling seeds, glumes, branches, knots, internodes, leaves and leaf sheaths;

3) and drying the sample and weighing. And (4) digesting the sample by using a graphite furnace, and measuring the molybdenum content of the sample by using ICP-MS.

This example shows the molybdate transporter encoding gene OsMOT1;2, the molybdenum content of the seeds, the molybdenum content of the glumes, the molybdenum content of the branches and stalks, the molybdenum content of the joints, the molybdenum content of the internodes, the molybdenum content of the leaves and the molybdenum content of the sheaths of the leaves of the mutants are all obviously reduced (figure 6).

Example 10

The molybdate transporter-encoding gene OsMOT1 prepared in example 4 under field conditions; 2, the molybdenum content of the over-expression strain is determined, and the specific implementation process is as follows:

1) contacting wild-type WT with OsMOT1;2 accelerating germination of seeds of the over-expression strain line at 37 ℃ for 2 days, sowing the seeds on a plastic black net suspended in tap water, culturing for two weeks, and transplanting seedlings to a field, wherein water and fertilizer management conforms to normal field management;

2) harvesting wild type and OsMOT1 in the mature period of rice; 2, sampling seeds, glumes, branches, knots, internodes, leaves and leaf sheaths of plants OE-1 and OE-2 of a super-expression strain;

3) and drying the sample and weighing. And (4) digesting the sample by using a graphite furnace, and measuring the molybdenum content of the sample by using ICP-MS.

This example shows the molybdate transporter encoding gene OsMOT1;2, the molybdenum content of the grains, the molybdenum content of the glumes, the molybdenum content of the branches, the molybdenum content of the joints and the molybdenum content of the internodes of the over-expression strain are all obviously improved (figure 7).

Sequence listing

<110> Nanjing university of agriculture

<120> application of encoding gene OsMOT1 of rice molybdate transporter and 2

<160> 20

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1786

<212> DNA

<213> Rice (Oryza sativa)

<400> 1

gtggatcaaa cttgagttca ctggactctg ccactacgga aatctggatc gcatcttaat 60

taagcctcga gagagacatt agtattttta ttcgctcatt ccaccggtca gaaacgtgtg 120

caagctaaca acatcgctag gcaaaaggaa gtcgttgctt tcgagtttcc cacccatggc 180

atcctccgcc ggcgacccgc tcctctccgg cgaggccggc gacggccgcc gcaggttcgt 240

cccgtccacc atacggctca agacgtcggt gtggtcggag ctgggcggcg cggtgggcga 300

tctgggcacc tacatcccca tcgtgctggc gctgtcgctc gcgtcccacc tcgacctcgg 360

caccacgctc atcttcaccg cgctctacaa cttcgccacc gggctcctct tcggcatccc 420

catgcccgtc cagcccatga agtccatcgc cgccgtcgcg ctctcctccg cgcacctcac 480

catcccgcag atcatgtccg ctggcctcgc cgtcgccgcc atcctcctct tcctcggcgt 540

caccggcctc atgaccaccc tctaccgcct cctcccgctc cccgtcgtgc gcggcgtcca 600

gctctcgcag ggcctctcct tcgccttcac cgccgtcaag tacatccgct acgtgcagga 660

cttctcccgt tcctcctctg cttccacctc cgtgccgcgc cccctcctcg gcctcgacgg 720

ccttgtcctc gcgctcgccg cgctactgtt cataatcctc gccaccggct ccggcgacga 780

cgaggacgtc aacagggacg gcacgagccg tcgccgtcgc tcctgcagcc gcgtcccggc 840

ggcgctaatc gtgttcgcgc tcggcttggt gctctgcttc gttcgtgatc cgtccatcct 900

gcaggatctc cgctttgggc cggcgccgtt ggggctggtc aagataacct gggacgattt 960

caagatcggg ttctgggagg gcgccgtgcc gcagctcccg ctgtccgtgc tgaactcggt 1020

gatcgccgtg tgcaagctgt cgtcggacct gttcccggaa cgggccgagc tctcgccggc 1080

gcgggtgtcg gtgagcgtgg ggctcatgaa tttcgtgggg tgctggttcg gcgccatgcc 1140

gtgctgccac ggcgcgggcg ggctggcggg gcagtaccgg ttcggcggcc ggaccggcgc 1200

gtccgtggtg ttcctggcca tcggcaagct ggcgctcggg ctggtgttcg gcaactcgtt 1260

cgtgacgatc ctggggcagt tcccgatcgg gatactgggc gtcatgctgc tcttctccgg 1320

gatcgagctc gccatggcgt cgcgcgacat ggggagcaag caggagtcgt tcgtcatgct 1380

ggtctgcgcc ggcgtgtcgc tcacaggctc gagcgccgcg ctgggcttca tctccggaat 1440

cgtgctgtac ctgttgctac gcctgaggga tttggagtgg gatatcagag gactgctcgg 1500

tcgctgggcc gcgggacggc ggcaatcgac caacgaggcc aatgaagatg gggctggaga 1560

tgcttgatcg aaatcttgag aacatctgtt gatttgagat tttgagtgtg gtgtgaattg 1620

ttcgagcgaa tatatatagt atcaacatgc atagcggata gccacgagtg attgaatttc 1680

tgaatggagt aactccacat aatcgcccaa actttcaaac caactactcc ctctgtcctt 1740

aaacatctta aatgtttgac gccgttggct tttttaaata tgtttg 1786

<210> 2

<211> 1392

<212> DNA

<213> Rice (Oryza sativa)

<400> 2

atggcatcct ccgccggcga cccgctcctc tccggcgagg ccggcgacgg ccgccgcagg 60

ttcgtcccgt ccaccatacg gctcaagacg tcggtgtggt cggagctggg cggcgcggtg 120

ggcgatctgg gcacctacat ccccatcgtg ctggcgctgt cgctcgcgtc ccacctcgac 180

ctcggcacca cgctcatctt caccgcgctc tacaacttcg ccaccgggct cctcttcggc 240

atccccatgc ccgtccagcc catgaagtcc atcgccgccg tcgcgctctc ctccgcgcac 300

ctcaccatcc cgcagatcat gtccgctggc ctcgccgtcg ccgccatcct cctcttcctc 360

ggcgtcaccg gcctcatgac caccctctac cgcctcctcc cgctccccgt cgtgcgcggc 420

gtccagctct cgcagggcct ctccttcgcc ttcaccgccg tcaagtacat ccgctacgtg 480

caggacttct cccgttcctc ctctgcttcc acctccgtgc cgcgccccct cctcggcctc 540

gacggccttg tcctcgcgct cgccgcgcta ctgttcataa tcctcgccac cggctccggc 600

gacgacgagg acgtcaacag ggacggcacg agccgtcgcc gtcgctcctg cagccgcgtc 660

ccggcggcgc taatcgtgtt cgcgctcggc ttggtgctct gcttcgttcg tgatccgtcc 720

atcctgcagg atctccgctt tgggccggcg ccgttggggc tggtcaagat aacctgggac 780

gatttcaaga tcgggttctg ggagggcgcc gtgccgcagc tcccgctgtc cgtgctgaac 840

tcggtgatcg ccgtgtgcaa gctgtcgtcg gacctgttcc cggaacgggc cgagctctcg 900

ccggcgcggg tgtcggtgag cgtggggctc atgaatttcg tggggtgctg gttcggcgcc 960

atgccgtgct gccacggcgc gggcgggctg gcggggcagt accggttcgg cggccggacc 1020

ggcgcgtccg tggtgttcct ggccatcggc aagctggcgc tcgggctggt gttcggcaac 1080

tcgttcgtga cgatcctggg gcagttcccg atcgggatac tgggcgtcat gctgctcttc 1140

tccgggatcg agctcgccat ggcgtcgcgc gacatgggga gcaagcagga gtcgttcgtc 1200

atgctggtct gcgccggcgt gtcgctcaca ggctcgagcg ccgcgctggg cttcatctcc 1260

ggaatcgtgc tgtacctgtt gctacgcctg agggatttgg agtgggatat cagaggactg 1320

ctcggtcgct gggccgcggg acggcggcaa tcgaccaacg aggccaatga agatggggct 1380

ggagatgctt ga 1392

<210> 3

<211> 37

<212> DNA