CN102220332B - Plant salt resistance associated miRNA (gma-miRN60) and use thereof - Google Patents
Plant salt resistance associated miRNA (gma-miRN60) and use thereof Download PDFInfo
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Abstract
The invention discloses a plant salt resistance associated miRNA, which has a sequence represented by SEQ ID No.1 in a sequence table. High-salt-resistance transgenic soybeans can be cultured by overexpressing the miRNA represented by SEQ ID No.1 or inhibiting the expression of the SEQ ID No.1 represented by miRNA in soybeans. The miRNA has a great significance for improving the yield of soybeans.
Description
Technical field
The present invention relates to a kind of miRNA relevant with plant salt endurance, especially a kind ofly derive from new miRNA soybean nodulation, that can regulate soybean salt-tolerance; The invention still further relates to the precursor of this miRNA, encoding gene of its precursor and uses thereof.
Background technology
MiRNA is the non-encoding histone RNA of endogenous of a kind of length 20～24 nt of Recent study discovery.The miRNA gene is transcribed at first and is produced the pre-miRNAs with neck ring structure.Pre-miRNA is transported out nuclear subsequently, by the effect of Dicer enzyme, is processed into ripe miRNA in tenuigenin.Be combined with the mRNA of particular target gene by sequence is complementary, thereby cause that mRNA degrades or inhibition mRNA translation is played down regulation (Llave et al., 2002 to the expression of gene; Palatnik et al., 2003; Tang et al., 2003).
The soil salinization is having a strong impact on growth and the growth of plant, is restricting agriculture production.Soybean is important in the world albumen and oil crops.Plant has a series of protection mechanism when the reply salt stress, the molecular mechanism of research Soybean Resistance salt, and cultivating the soybean salt-tolerance new variety is to improve a main path of soybean yields.At present, in the plant anti-salt Mechanism Study, research comparatively is clear that SOS approach (Qiu et al., 2002; Quintero et al., 2002), also be cloned into relevant gene (Li et al., 2005 of some anti-salt in the soybean; Liao et al., 2008; Xie et al., 2009; Cheng et al., 2009).In addition, there are some researches show that (Zhu et al., 2004 microRNA(miRNA) also play a significant role in plant anti-salt mechanism; Jung et al., 2007; Zhao et al., 2009).
Summary of the invention
The encoding gene that the technical problem to be solved in the present invention provides a kind of miRNA, its precursor sequence and precursor sequence thereof relevant with plant salt endurance utilizes this miRNA can improve the salt tolerance of soybean, is significant to improving soybean yields.
For solving the problems of the technologies described above, the technical solution used in the present invention is as follows.
A kind of miRNA relevant with plant salt endurance, it has the sequence shown in the SEQ ID NO:1 in the sequence table.
The precursor sequence of the above-mentioned miRNA relevant with plant salt endurance, it has the sequence shown in the SEQ ID NO:2 in the sequence table.
The dna sequence dna of the above-mentioned miRNA precursor sequence relevant with plant salt endurance of encoding, it has the sequence shown in the SEQ ID NO:3 in the sequence table.
Above-mentioned miRNA the application during cultivating salt tolerant transgenic plant relevant with plant salt endurance.
As a kind of optimal technical scheme of above-mentioned application, described salt tolerant transgenic plant are soybean.
As a kind of optimal technical scheme of above-mentioned application, in soybean, cross and express the miRNA shown in the SEQ ID NO:1, or suppress the expression of miRNA shown in the SEQ ID NO:1, cultivate the genetically engineered soybean with high-salt tolerance.
The beneficial effect that adopts technique scheme to produce is: the invention provides a kind of new soybean miRNA, Solexa order-checking and real-time fluorescence quantitative PCR detected result show, the expression of miRNA of the present invention is subject to the adjusting of salt stress, illustrate that it plays an important role in soybean adaptation high-salt stress mechanism, based on this, can utilize its cultivation to have the genetically engineered soybean of high-salt tolerance, be significant to improving soybean yields.
Description of drawings
Express number of times under Fig. 1 salt stress that is gma-miRN60 in the Solexa order-checking and the non-condition of salt stress, the result shows, gma-miRN60 expresses number under the normal growth condition be 12, expressing number of times after salt is processed is 66, show that the expression of gma-miRN60 is subject to the adjusting of salt stress, illustrate that it plays an important role in soybean adaptation high-salt stress mechanism.
Fig. 2 is the loop-stem structure figure of gma-miRN60 precursor sequence, and as seen its precursor sequence is folded into a kind of stable loop-stem structure, belongs to the typical secondary structure of miRNA precursor, meets the constitutional features of miRNA precursor.
Fig. 3 is real-time fluorescence quantitative PCR to the analytical results of gma-miRN60 at expression characteristic under the condition of salt stress and under the non-condition of salt stress, the result shows that the expression of gma-miRN60 is subjected to salt to induce obvious rise, illustrates that it plays an important role in soybean adaptation high-salt stress mechanism.
Embodiment
Following examples describe the present invention in detail.Various raw material used in the present invention and items of equipment are conventional commercially available prod, all can buy directly by market to obtain.Quantitative test in following examples all arranges repeated experiments three times, results averaged.
MiRNA provided by the invention derives from the soybean mature nodule, called after gma-miRN60, and its length is 21nt; Referring to accompanying drawing 2, its precursor sequence can be folded into a kind of stable loop-stem structure, belongs to the typical secondary structure of miRNA precursor, meets the constitutional features of miRNA precursor.
The effect of embodiment 1:Solexa sequencing analysis checking gma-miRN60 in soybean salt-tolerance mechanism
One, material is cultivated and the NaCl processing:
Monkey hair soybean seeds is with 70% alcohol, 30 S that sterilize, dH
2O is seeded in the sterile petri dish after washing 6 times, puts 2 aseptic filter papers at the bottom of the ware, and dH2O soaks, 28 ℃ of dark sproutings 3 days; When bud grows to 2 cm, move in the test tube that is placed with moistening filter paper, bud grows to 4～5 cm; move in the root nodule bacterium bacterium liquid of activation, inoculate 30 min, low N water planting; 10,000 lx, temperature is 26 ℃; relative humidity is 70%, cultivates 28 days, and 125 mM NaCl processed 5 hours; contrast is home; get root nodule, liquid nitrogen freezing ,-80
oThe C storage.
Two, Solexa sequencing analysis:
Upper step gained sample is sent to the large genome company of China, carry out solexa order-checking and analysis, referring to Fig. 1, the result shows, gma-miRN60 expresses number under the normal growth condition be 12, expressing number of times after salt is processed is 66, this shows that the expression of gma-miRN60 is subject to the adjusting of salt stress, illustrate that it plays an important role in soybean adaptation high-salt stress mechanism, in soybean, cross expression gma-miRN60, or suppress the expression of gma-miRN60, will affect the adaptive faculty of soybean to salt stress, cultivate the genetically engineered soybean with high-salt tolerance, the output that improves soybean is significant.
Embodiment 2: the effect of real-time fluorescence quantitative PCR (Real-time PCR) analysis verification gma-miRN60 in soybean salt-tolerance mechanism
One, material is cultivated and the NaCl processing:
Monkey hair soybean seeds is with 70% alcohol, 30 S that sterilize, dH
2O is seeded in the sterile petri dish after washing 6 times, puts 2 aseptic filter papers at the bottom of the ware, and dH2O soaks, 28 ℃ of dark sproutings 3 days; When bud grows to 2 cm, move in the test tube that is placed with moistening filter paper, bud grows to 4～5 cm; move in the root nodule bacterium bacterium liquid of activation, inoculate 30 min, low N water planting; 10,000 lx, temperature is 26 ℃; relative humidity is 70%, cultivates 28 days, and 125 mM NaCl processed 5 hours; contrast is home; get root nodule, liquid nitrogen freezing ,-80
oThe C storage;
Two, the separation of miRNA is extracted test kit (hundred Imtech, Cat # RP5301) sampling with microRNA and is extracted small fragment RNA, and extracting method is as follows:
(1) homogenized: the root nodule of getting grinds in mortar fast with liquid nitrogen, and per 50～100 mg tissue adds homogenate behind the lysate in the 1 ml test kit;
(2) with homogenate sample concuss mixing, under 15～30 ℃ of conditions, hatch 5 min so that ribosome decomposes fully:
(3) centrifugal 10 min of 12,000 rpm under 4 ℃ condition carefully get in the centrifuge tube that supernatant changes a new RNase free over to;
(4) per 1 ml lysate adds 0.2 ml chloroform; Cover tightly sample hose lid, thermal agitation 15s is also at room temperature hatched 3min with it;
(5) sample can be divided into three layers in 4 ℃, centrifugal 10 min of 12,000 rpm: lower floor's organic phase, and the water that middle layer and upper strata are colourless, RNA is present in aqueous phase; The capacity of aqueous phase layer be approximately add 60% of lysate volume, water is transferred in the new pipe, carry out next step operation;
(6) add 0.6 times of volume 70% ethanol, put upside down mixing (precipitation may appear in this moment); The solution that obtains changes among the interior adsorption column RA of microRNA extraction test kit together with precipitating;
(7) 10, centrifugal 45 s of 000 rpm, collect lower filtrate (containing miRNA in the lower filtrate), accurately estimate the volume of lower filtrate, add the dehydrated alcohol of 2/3 times of volume, put upside down several times mixing, mixed solution is poured into microRNA to be extracted among the adsorption column RB in the test kit, centrifugal 30 s of 10,000 rpm discard waste liquid;
(8) add the interior rinsing liquid RW of 700 μ l test kits, centrifugal 60 s of 12,000 rpm discard waste liquid;
(9) add the interior rinsing liquid RW of 500 μ l test kits, centrifugal 60 s of 12,000 rpm discard waste liquid;
(10) adsorption column RB is put back in the sky collection tube, centrifugal 2 min of 12,000 rpm remove rinsing liquid as far as possible, in order to avoid residual ethanol suppresses downstream reaction in the rinsing liquid;
(11) take out adsorption column RB, put into a RNase free centrifuge tube, add 60～80 μ l RNase free water(in the adsorption film middle part and in 65～70 ℃ of water-baths, heat in advance), room temperature is placed 2 min, then centrifugal 1 min of 12,000 rpm; Collection obtains pure microRNA and is stored in-80 ℃ of refrigerators;
Three, reverse transcription is cDNA, utilizes day root miRNA cDNA the first chain synthetic agent box that the miRNA reverse transcription of purifying is cDNA:
(1) Poly of miRNA (A) tailing, the reaction solution prescription is (each composition except miRNA is all taken from the mentioned reagent box): miRNA, 6 μ l; E-PAP(5U/ μ l), 0.4 μ l; 10 * PAP Buffer, 2 μ l; 5 * rATP solution, 4 μ l; Nuclease-free Water, 7.6 μ l; Cumulative volume, 20 μ l; The reaction solution of the above-mentioned preparation of mixing is of short duration centrifugal so that the reaction solution on the tube wall sinks 37 ℃ of reaction 60min gently; The gained reaction solution can directly carry out the downstream experiment, also can be placed on-20 ℃ of of short duration preservations, deposits in-80 ℃ such as the need prolonged preservation;
(2) miRNA of Poly (A) modification carries out reverse transcription reaction, and the reaction solution prescription is (each composition except Poly (A) reaction solution is all taken from test kit): Poly (A) reaction solution, 2 μ l; 10 * RT primer, 2 μ l; 10 * RT Buffer, 2 μ l; Ultrapure dNTP Mixture (2.5Mm), 1 μ l; Rnasin (40U/ μ l), 1 μ l; Quant Rtase, 0.5 μ l; RNase-free ddH
2O, 1.5 μ l; Cumulative volume, 20 μ l; The reaction solution of the above-mentioned preparation of mixing is of short duration centrifugal so that the reaction solution on the tube wall sinks 37 ℃ of reaction 60min gently; Synthetic cDNA reaction solution is placed-20 ℃ of preservations, also can directly carry out the downstream fluorescent quantitation and detect;
Four, real-time fluorescence quantitative PCR:
Then the following reaction system of mixing is distributed in the 96 hole optical sheets, and covers upper blooming, and the instrument that amplification is used is the quantitative real time PCR Instrument of ABI company;
Amplification program is: 95 ℃ of 30s; 95 ℃ of 5 s, 60 ℃ of 34s, 45 cycles; 95 ℃ of 15 s, 60 ℃ of 1min, 95 ℃ of 15 s;
20 μ l reaction systems are formulated as follows: dna profiling, 2 μ l; SYBR
Primix Ex taq TM (2 *), 10 μ l; PCR Forward Primer(10 μ Μ), 0.4 μ l; PCR Reverse Primer(10 μ Μ), 0.4 μ l; ROX Reference Dye II (50 *), 0.4 μ l; DdH
2O, 6.8 μ l; Cumulative volume, 20 μ l;
Primer sequence is: N60-F:AGCCGCGTCAATATCTTATTT(SEQ ID NO:4); 5.8S rRNA-F:ACGCCTGCCTGGGTGTCACAC(SEQ ID NO:5); AMRM:GCGAGCACAGAATTAATACGACT(SEQ ID NO:6); (5.8S rRNA is confidential reference items, and AMRM is reverse primer);
Five, result:
Referring to Fig. 3, the result of real-time fluorescence quantitative PCR analysis shows that the expression of gma-miRN60 is subjected to salt to induce obvious rise, this shows that equally the expression of gma-miRN60 is subject to the adjusting of salt stress, in soybean, cross expression gma-miRN60, or the expression of inhibition gma-miRN60, with affecting the adaptive faculty of soybean to salt stress, cultivate the genetically engineered soybean with high-salt tolerance, the output that improves soybean is significant.
Foregoing description only proposes as the enforceable technical scheme of the present invention, not as the Single restriction condition to its technical scheme itself.
Sequence table
<160> 6
<210> 1
<211> 21
<212> RNA
＜213〉Glycine soybean (Glycine max)
<400> 1
AGCCGCGUCA AUAUCUUAUU U 21
<210> 2
<211> 109
<212> RNA
＜213〉Glycine soybean (Glycine max)
<400> 2
ACAUGACAAA UUAAGGUAUU GGCGUGCCUC AAUUUGAAUA CAUGGCUAUU AUGACAAAUC 60
CAGCCUUGUA GUUUGAUUGA GCCGCGUCAA UAUCUUAUUU UGCUCUUCU 109
<210> 3
<211> 109
<212> DNA
＜213〉Glycine soybean (Glycine max)
<400> 3
ACATGACAAA TTAAGGTATT GGCGTGCCTC AATTTGAATA CATGGCTATT ATGACAAATC 60
CAGCCTTGTA GTTTGATTGA GCCGCGTCAA TATCTTATTT TGCTCTTCT 109
<210> 4
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 4
AGCCGCGTCA ATATCTTATT T 21
<210> 5
<211> 21
<212> DNA
＜213〉artificial sequence
<400> 5
ACGCCTGCCT GGGTGTCACA C 21
<210> 6
<211> 23
<212> DNA
＜213〉artificial sequence
<400> 6
GCGAGCACAG AATTAATACG ACT 23
Claims (3)
1. miRNA relevant with plant salt endurance, it is characterized in that: its sequence is shown in SEQ ID NO:1.
2. the precursor sequence of the miRNA relevant with plant salt endurance claimed in claim 1, it is characterized in that: its sequence is shown in SEQ ID NO:2.
3. the dna sequence dna of the miRNA precursor sequence relevant with plant salt endurance claimed in claim 2 of encoding, it is characterized in that: its sequence is shown in SEQ ID NO:3.
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Non-Patent Citations (6)
| Title |
|---|
| Identification of novel and candidate miRNAs in rice by high throughput sequencing;Sunkar et al;《BMC Plant Biology》;20080229;第25卷(第8期);pp.1-17 * |
| MicroRNAs in the shoot apical meristem of soybean;Wong et al;《Journal of Experimental Botany》;20110419;第62卷(第8期);pp.2495–2506 * |
| Sunkar et al.Identification of novel and candidate miRNAs in rice by high throughput sequencing.《BMC Plant Biology》.2008,第25卷(第8期),pp.1-17. |
| Wong et al.MicroRNAs in the shoot apical meristem of soybean.《Journal of Experimental Botany》.2011,第62卷(第8期),pp.2495–2506. |
| 耐旱野生大豆MicroRNA的鉴定与表达分析;陈锐;《中国博士学位论文全文数据库 农业科技辑》;20091015(第10期);全文 * |
| 陈锐.耐旱野生大豆MicroRNA的鉴定与表达分析.《中国博士学位论文全文数据库 农业科技辑》.2009,(第10期),全文. |
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