CN116219052B - Marker gene Pt7G61650 for identifying different periods of nutrient growth of slash pine and application method thereof - Google Patents
Marker gene Pt7G61650 for identifying different periods of nutrient growth of slash pine and application method thereof Download PDFInfo
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Abstract
The invention provides a marker gene Pt7G61650 for identifying different periods of nutrient growth of slash pine and an application method thereof, and relates to the technical field of molecular biology detection. The marker gene Pt7G61650 can identify different periods of nutrient growth of the slash pine, and the sequence of the gene is shown as SEQ ID NO. 1. The invention analyzes the expression quantity of Pt7G61650 gene in the slash pine tissue by qRT-PCR technology through micro-sampling of tree tissue, efficiently and accurately identifies different periods of nutrition growth (namely nutrition growth juvenile period or nutrition growth adult period) of the slash pine plant and even selected branches, and provides guidance for forestry production and stand management.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to a marker gene Pt7G61650 for identifying different periods of nutrient growth of slash pine and an application method thereof.
Background
Pine is one of the most important industrial wood species in the world, and Pinus elliottii x p.caribaea is an important economic and artificial forest pine species in the south of China. Since 90 s of the last century, guangdong province began the work of wet pine breeding and clone breeding, wet pine was widely planted as a fast-growing and productive forest in Australia and America, with wet pine (P.elliottii) as a female parent and Caribaea (P.caribaea) as a male parent.
The higher plant needs to go through a vegetative growth stage (reproductive stage) and a reproductive growth stage (v) after the seeds germinate, when the plant grows to a certain period in nutrition, the plant can bloom and fruit to enter the reproductive stage, the vegetative growth stage can be further divided into two different periods, namely a juvenility period and an adult stage, and in forestry production practice, the slash needs to take branches in the juvenility period as materials to carry out asexual forestation so as to enable the offspring to survive and keep good growth vigor; and materials entering the reproductive phase are needed in the production of forests. However, there have long been difficulties in determining the time of transition of trees, especially pine, from the juvenile period to the adult period, and thus, in vivo assays have not been possible, and thus, using molecular biological detection techniques, it is critical to accurately identify the developmental stage of pine plants, and even selected shoots, in a living state to improve forestry production efficiency.
Disclosure of Invention
Therefore, the invention aims to identify the marker gene Pt7G61650 of the slash pine in different periods of nutrient growth and an application method thereof, and the gene can be used for identifying the different periods of nutrient growth of the slash pine and provides guidance for forestry production and stand management.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a marker gene Pt7G61650 for identifying different periods of nutrient growth of slash pine, wherein the nucleotide sequence of the marker gene Pt7G61650 is shown as SEQ ID NO. 1.
Preferably, the different periods of vegetative growth of the slash pine include vegetative growth juvenile period and vegetative growth adult period;
when the slash pine is in the vegetative growth juvenile period, the tree does not bloom or can maintain the flowering capacity, the top meristem of the branch does not have sexual reproduction capacity, the branch has stronger adventitious root generation capacity, and the slash pine is suitable for asexual propagation such as cuttage;
when the slash pine is in the vegetative growth adult stage, the tree obtains the flowering capacity or potential, the top meristem of the branch loses the nutrition state, the branch loses the adventitious root generating capacity, and the slash pine is suitable for character selection, wood property measurement and the like.
The invention also provides application of the marker gene Pt7G61650 in identifying different periods of nutrient growth of the slash pine.
The invention also provides a method for identifying different periods of nutrient growth of the slash pine, which comprises the following steps: extracting total RNA of a tissue to be detected of the slash pine and a control sample, and respectively measuring the expression quantity of the marker gene Pt7G61650 after reverse transcription;
if the control sample is a sample of 0.5-5 years old wet-added pine: when the expression quantity of the marker gene Pt7G61650 of the tissue sample to be detected has no obvious difference, the tissue to be detected is in the vegetative growth juvenile period; when the expression quantity of the marker gene Pt7G61650 of the tissue sample to be detected is 2 times larger than that of the control sample, the tissue to be detected is in vegetative growth adult phase.
If the control sample is a sample of greater than 6.5 years old wet plus pine: when the expression level of the tissue sample marker gene Pt7G61650 to be detected is within one half of the expression level of the control sample, the tissue to be detected is in the vegetative growth juvenile period; when the expression level of the tissue sample marker gene Pt7G61650 to be detected is not obviously different from that of the control sample, the tissue to be detected is in vegetative growth adult phase.
Preferably, the primers used for determining the expression level comprise an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.3.
Preferably, the control samples include a vegetative growth juvenile control sample and a vegetative growth adult control sample.
Preferably, the control sample comprises tender stems of the lowest layer of branches of 0.5-5 years old wet-added pine, which are 10 cm-12 cm away from the tips.
Preferably, the control sample comprises tender stems in the lowest layer of branches of 10 years old wet-plus-pine at a distance of 10cm to 12cm from the tip.
Preferably, the method also comprises an internal reference Pt18S when the expression quantity is measured, and the nucleotide sequence of a primer pair of the internal reference Pt18S is shown as SEQ ID NO.4 and SEQ ID NO. 5.
Preferably, the expression level is determined by qRT-PCR, and the procedure of the qRT-PCR comprises: pre-denaturation at 95℃for 2 min; PCR reactions at 94℃for 5s, 60℃for 30s,39 cycles; melting curve analysis at 65-95℃increased by 0.5℃every 5 s.
The invention also provides a kit for identifying different periods of nutrient growth of the slash pine, which comprises a specific primer pair designed for the marker gene Pt7G61650, wherein the specific primer pair comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.3.
The beneficial effects are that: the invention provides a marker gene Pt7G61650 for identifying different periods of nutrient growth of slash pine, which can identify different periods of nutrient growth of slash pine by utilizing the Pt7G61650 gene, and the sequence of the gene is shown as SEQ ID NO. 1. The invention analyzes the expression quantity of Pt7G61650 gene in the slash pine tissue by qRT-PCR technology through micro-sampling of tree tissue, efficiently and accurately identifies different periods (namely juvenile period or adult period) of nutrition growth of the slash pine plant and even selected branches, and provides guidance for forestry production and stand management.
Drawings
FIG. 1 shows the Pt7G61650 gene expression level in the lowest shoots of 0.5 years, 2 years, 4 years, 6.5 years and 10 years of wet-added pine;
FIG. 2 shows the Pt7G61650 gene expression level in the sample to be tested and the control sample.
Detailed Description
The invention provides a marker gene Pt7G61650 for identifying different periods of nutrient growth of slash pine, wherein the nucleotide sequence of the marker gene Pt7G61650 is shown as SEQ ID NO. 1.
The marker gene Pt7G61650 is preferably derived from the slash pine, and the expression level of the marker gene Pt7G61650 shows extremely remarkable difference in different periods of the vegetative growth of the slash pine, so that the marker gene Pt7G61650 can be used for distinguishing the juvenile period of the vegetative growth from the adult period of the vegetative growth. In the present invention, the vegetative growth juvenile period is: when the tree is in the period, the capability of flowering or flowering maintenance is avoided, the top meristem of the branch has no sexual reproduction capability, the branch has stronger adventitious root generation capability, and the method is suitable for asexual propagation such as cuttage and the like; the vegetative growth adult phase is as follows: when the tree is in the period, the flowering capacity or potential is obtained, the top meristem of the branch loses the nutrition state, the branch loses the adventitious root generating capacity, and the method is suitable for character selection, material property measurement and the like. In production practice and related studies, most conifer trees in vegetative growth juvenile stage are less than 6.5 years old, and most conifer trees in vegetative growth juvenile stage are more than 6.5 years old. This was experimentally verified in step 3) of example 1 of the present invention.
The invention also provides application of the marker gene Pt7G61650 in identifying different periods of nutrient growth of the slash pine.
The invention also provides a method for identifying different periods of nutrient growth of the slash pine, which comprises the following steps: extracting total RNA of a tissue to be detected of the slash pine and a control sample, and respectively measuring the expression quantity of the marker gene Pt7G61650 after reverse transcription;
if the control sample is a sample of 0.5-5 years old wet-added pine: when the expression quantity of the marker gene Pt7G61650 of the tissue sample to be detected has no obvious difference, the tissue to be detected is in the vegetative growth juvenile period; when the expression quantity of the marker gene Pt7G61650 of the tissue sample to be detected is 2 times larger than that of the control sample, the tissue to be detected is in vegetative growth adult phase.
If the control sample is a sample of greater than 6.5 years old wet plus pine: when the expression level of the tissue sample marker gene Pt7G61650 to be detected is within one half of the expression level of the control sample, the tissue to be detected is in the vegetative growth juvenile period; when the expression level of the tissue sample marker gene Pt7G61650 to be detected is not obviously different from that of the control sample, the tissue to be detected is in vegetative growth adult phase.
The method for extracting the RNA is not particularly limited, and preferably, the Trizol method is used for extracting total RNA of a sample, and when the RNA of a tissue or the sample is extracted, sampling is carried out according to the extractable amount of the sample, and finally, the total RNA can be extracted and applied to qRT-PCR. The tissue to be detected preferably comprises different wet-added pine or branches at different positions of the same wet-added pine, so that different periods of nutrient growth of different wet-added pine can be identified based on the method disclosed by the invention, and the tissue to be detected can be even used for identifying different periods of nutrient growth of different positions of the same tree, and particularly comprises different periods of nutrient growth of organs or tissues such as wet-added pine needles, branches, cambium and the like.
The control sample of the present invention preferably comprises wet-added pine of known age and in vegetative growth juvenile phase, and wet-added pine of known age and in vegetative growth juvenile phase; wherein the control sample in the vegetative growth juvenile period is more preferably obtained from tender stems 10cm to 12cm away from the tip in the lowest layer of branches of 0.5 to 5 years old wet plus pine, and the control sample in the vegetative growth juvenile period is more preferably obtained from tender stems 10cm to 12cm away from the tip in the lowest layer of branches of 10 years old wet plus pine.
The invention carries out reverse transcription on total RNA extracted from the tissue to be detected and the control sample respectively, and the method for carrying out reverse transcription is not particularly limited, and can be carried out by utilizing a conventional kit in the field. The cDNA obtained by reverse transcription is used as a template, the expression quantity of the marker gene Pt7G61650 is determined by adopting a qRT-PCR method, the primers used for determining the expression quantity preferably comprise an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is preferably shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is preferably shown as SEQ ID NO.3. In the invention, when the expression quantity is measured, the primer pair nucleotide sequence of the internal reference Pt18S is preferably shown as SEQ ID NO.4, and the primer pair nucleotide sequence of the internal reference Pt18S is preferably shown as SEQ ID NO. 4: CGGCTACCACATCCAAGGAA, and SEQ ID NO. 5: GCTGGAATTACCGCGGCT.
The invention utilizes qRT-PCR to measure the expression quantity, the qRT-PCR is preferably carried out by adopting a two-step method for reaction, and the program is preferably designed as follows: pre-denaturation at 95℃for 2 min; PCR reactions at 94℃for 5s, 60℃for 30s,39 cycles; melting curve analysis reaction was performed at 65-95℃with an increase of 0.5℃every 5 s. The expression level according to the invention is preferably based on 2 -ΔΔCt The relative expression calculated by the method is as the reference sample is 0.5-5 years real wet plus pine sample: when the expression quantity of the marker gene Pt7G61650 of the tissue sample to be detected has no obvious difference, the tissue to be detected is in the vegetative growth juvenile period; when the expression quantity of the marker gene Pt7G61650 of the tissue sample to be detected is 2 times larger than that of the control sample, the tissue to be detected is in vegetative growth adult phase. If the control sample is a sample of greater than 6.5 years old wet plus pine: when the expression level of the tissue sample marker gene Pt7G61650 to be detected is within one half of the expression level of the control sample, the tissue to be detected is in the vegetative growth juvenile period; when the expression level of the tissue sample marker gene Pt7G61650 to be detected is not obviously different from that of the control sample, the tissue to be detected is in vegetative growth adult phase.
The invention also provides a kit for identifying different periods of nutrient growth of the slash pine, which comprises a specific primer pair designed for the marker gene Pt7G61650, wherein the specific primer pair comprises an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.3.
The kit of the invention preferably also includes Pt18S, as well as other conventional reagents for performing qRT-PCR, such as TransStart Top Green qPCR SuperMix and RNase-Free Water.
The marker gene Pt7G61650 for identifying different periods of nutrient growth of slash pine and the application method thereof provided by the invention are described in detail below with reference to examples, but they are not to be construed as limiting the scope of the invention.
Example 1
1) Reference sample collection
Collecting the lowest layer of tip branches of wet pine of 0.5 years, 2 years, 4 years, 6.5 years and 10 years in a red-mountain seed garden (112 DEG 49'N,22 DEG 9'W) of Taishan city, guangdong province, taking tender stem segments of the collected branches, which are 10 cm-12 cm away from the tip, as sample materials, immediately freezing the branches with liquid nitrogen after collection, and storing the branches in an ultralow temperature refrigerator at-80 ℃.
2) Transcriptome sequencing and analysis of reference samples
Total RNA of the reference sample is extracted by a Trizol method, and is subjected to quality inspection, a NEB #7530 kit (#E7530, new England Biolabs) is used for transcriptome library construction, and after the library quality inspection is qualified, the library is sent to Guangzhou Diao biotechnology Co., ltd, and high-throughput sequencing is carried out on an Illumina nova-6000 platform.
The expression level of Pt7G61650 gene was characterized by the normalization method of TPM (Transcript per million) using the high quality genome Chinese pine genome (https:// www.ncbi.nlm.nih.gov/biopjet/PRJNA 784915) of the closely related species Pinus radiata (Pinus tabuliformis) of Pinus radiata as a reference genome, and data visualization was achieved (FIG. 1).
3) Age interval setting for control samples
As shown in FIG. 1, the average value of TPM of Pt7G61650 gene in 3 biological repeated samples was 165.1, 99.5, 110.4 and 125.1 in the 0.5, 2, 4 and 6.5 year tree-age wet-added pine samples in the young period, and the average value of TPM of Pt7G61650 gene in 3 biological repeated samples was 547.7 in the 10 year tree-age wet-added pine samples in the adult period. The result reflects that the expression quantity of Pt7G61650 gene is extremely obvious in the nutrition growth juvenile period and the nutrition growth adult period of the wet pine, the gene can effectively reflect and identify different nutrition growth periods of the wet pine, so that the lowest branch of the wet pine with the age of 0.5-6.5 years can be used as a control sample of the nutrition growth juvenile period, and the lowest branch of the wet pine with the age of 10 years can be used as a control sample of the nutrition growth adult period.
4) Primer design
According to CDS sequence of age marker gene PtAP2L3, designing primers of NCBI on-line Primer design website Primer-BLAST, wherein the Primer sequences are SEQ ID NO.2 and SEQ ID NO.3.
5) Sample to be tested and control sample collection
Collecting branches of a higher canopy layer of wet pine in an artificial forest, and taking tender stem segments 10 cm-12 cm away from the tip as a tested sample; meanwhile, the lowest layer of branches of 10-year-old wet pine with known ages is collected, and tender stem segments with the distance of 10 cm-12 cm from the tips are taken as a control sample material. The materials are quickly frozen by liquid nitrogen after being collected, and are stored in an ultralow temperature refrigerator at the temperature of minus 80 ℃.
6) Extraction of total RNA from a sample to be tested and a control sample
And (3) extracting total RNA of the tested sample and the control sample collected in the step 5) by using an RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit (DP 441) of Tiangen biochemical technology (Beijing) limited company. RNA concentration and integrity were determined by Nano-micro spectrophotometry and 1% agarose gel electrophoresis, respectively.
7) Reverse transcription of total RNA of test sample and control sample
In an RNase free PCR tube, the reverse transcription reaction of the total RNA obtained in step 6) was completed, and the genomic DNA was removed. Reverse transcription systems were prepared according to Table 1, the prepared systems were gently mixed, incubated in a PCR apparatus at 42℃for 15min, then heated at 85℃for 5s to completely inactivate RT/RI enzymes and gDNA reverse, and the products were stored at-20 ℃.
TABLE 1 reverse transcription system
8) Real-time fluorescent quantitative PCR (qRT-PCR) detection of Pt7G61650 gene
Diluting the cDNA generated by reverse transcription in the step 7) 70 times, taking the diluted cDNA as a template, carrying out a qRT-PCR experiment according to a qRT-PCR reaction system and reaction conditions shown in the table 2, and adopting a SYBR Green chimeric fluorescence method to carry out reaction, wherein a pre-denaturation reaction program is set to be 2min at 95 ℃, and a PCR reaction program is set to be 5s at 94 ℃, 30s at 60 ℃ and 39 cycles; the melting curve analysis reaction procedure was set to 65-95 ℃ with an increase of 0.5 ℃ every 5 s. Each sample was subjected to 3 technical replicates and 3 biological replicates, and independent reference Pt18S was added to each plate sample.
Finally, the expression level of Pt7G61650 gene in the sample to be tested and the control sample is obtained (figure 2).
TABLE 2 qRT-PCR reaction System
9) Identification of different periods of nutrient growth of tested sample
And judging different periods of vegetative growth of the tested sample according to the expression quantity of the Pt7G61650 gene after the real-time fluorescence quantitative PCR of the tested sample and the control sample, wherein the result is shown in figure 2. The control sample is a 10-year-old sample in the vegetative growth adult stage, and the expression level of the Pt7G61650 gene in the sample to be detected is less than one half of the expression level of the control sample, so that the vegetative growth stage of the sample to be detected is the vegetative growth juvenile stage.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. Marker gene for identifying different periods of nutrient growth of slash pinePt7G61650Characterized in that the marker genePt7G61650The nucleotide sequence of (2) is shown as SEQ ID NO. 1.
2. The marker gene according to claim 1Pt7G61650Wherein the different periods of nutrient growth of the slash pine comprise the young growth of the nutrient growthStage and vegetative growth adult stage;
when the slash pine is in the vegetative growth juvenile period, the tree does not bloom or can maintain the flowering capacity, the top meristem of the branch does not have sexual reproduction capacity, and the branch has stronger adventitious root generation capacity;
when the slash is in the vegetative growth adult stage, the tree obtains the ability or potential of flowering, the top meristem of the branch loses the nutritional state, and the adventitious root of the branch has weak occurrence ability.
3. The marker gene according to claim 1 or 2Pt7G61650Use in identifying different periods of nutrient growth of slash pine.
4. A method for identifying different periods of nutrient growth of slash pine, comprising the steps of: extracting total RNA of a tissue to be tested of the slash pine and a control sample, and measuring the marker genes according to claim 1 or 2 respectively after reverse transcriptionPt7G61650Expression level of (2);
when the control sample is a sample of 0.5-5 years of real wet pine, the marker genes of the tissue to be detected and the control samplePt7G61650The tissue to be tested is in the vegetative growth juvenile period if the expression level of the tissue to be tested is not significantly different; when the tissue to be tested is marked with genesPt7G61650The expression quantity of the tissue to be detected is 2 times greater than that of a control sample, and the tissue to be detected is in vegetative growth adult phase;
when the control sample is a 10-year-old wet-added pine sample, the tissue marker gene to be detectedPt7G61650The expression level of the test sample is less than half of the expression level of the control sample, and the tissue to be tested is in the vegetative growth juvenile period; when the tissue to be tested is marked with genesPt7G61650The expression level of the test sample is not obviously different from that of a control sample, and the tissue to be tested is in vegetative growth adult phase.
5. The method according to claim 4, wherein the primers used for determining the expression level comprise an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.3.
6. The method of claim 4, wherein the control sample comprises tender stems from 10cm to 12cm distal to the tip of a lowest layer of shoots of 0.5-5 year old wet-plus-pine.
7. The method of claim 4 or 6, wherein the control sample further comprises tender stems in the lowest layer of 10-year-old wet-plus-pine shoots at a distance of 10cm to 12cm from the tips.
8. The method according to claim 4, wherein the method further comprises the step of measuring the expression level of an internal referencePt18SThe internal referencePt18SThe nucleotide sequences of the primer pairs are shown as SEQ ID NO.4 and SEQ ID NO. 5.
9. The method of claim 4, wherein the amount of expression is determined using qRT-PCR, and wherein the procedure of qRT-PCR comprises: 95. pre-denaturation at 2 min; 94. PCR reactions at 5℃ 5s, 60℃for 30s,39 cycles; melting curve analysis reaction is carried out at 65-95 ℃ with 0.5 ℃ added every 5. 5 s.
10. A kit for identifying different periods of nutrient growth of slash pine, comprising a marker gene according to claim 1 or 2Pt7G61650The designed specific primer pair comprises an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO.3.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101027993A (en) * | 2006-09-14 | 2007-09-05 | 厦门涌泉科技有限公司 | Growth regulator for wet canada pine cuttage and its use |
| KR20080101883A (en) * | 2008-07-28 | 2008-11-21 | 세레스 인코퍼레이티드 | Nucleotide sequences and corresponding polypeptides conferring modulated plant growth rate and biomass in plants |
| CN102812893A (en) * | 2012-08-14 | 2012-12-12 | 广东省林业科学研究院 | Culture method for rooting slash pine x Caribbean pine F1 hybrid needle fascicles |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6921643B2 (en) * | 2003-02-05 | 2005-07-26 | The Regents Of The University Of California | Compositions and methods for detecting a sequence mutation in the cinnamyl alcohol dehydrogenase gene associated with altered lignification in loblolly pine |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101027993A (en) * | 2006-09-14 | 2007-09-05 | 厦门涌泉科技有限公司 | Growth regulator for wet canada pine cuttage and its use |
| KR20080101883A (en) * | 2008-07-28 | 2008-11-21 | 세레스 인코퍼레이티드 | Nucleotide sequences and corresponding polypeptides conferring modulated plant growth rate and biomass in plants |
| CN102812893A (en) * | 2012-08-14 | 2012-12-12 | 广东省林业科学研究院 | Culture method for rooting slash pine x Caribbean pine F1 hybrid needle fascicles |
Non-Patent Citations (1)
| Title |
|---|
| Genetic control of adventitious rooting on stem cuttings in two Pinus elliottii × P. caribaea hybrid families;Mervyn SHEPHERD 等;Ann. For. Sci;第62卷;第403–412页 * |
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