Application of marker gene PtAP2R1 in identifying vegetative growth stage and reproductive growth stage of Chinese pines

Abstract

The invention belongs to the technical field of plant gene engineering, and particularly relates to application of a marker gene PtAP2R1 to identification of a vegetative growth stage and a reproductive growth stage of Chinese pines. The marker gene PtAP2R1 is massively expressed in the vegetative growth stage of the Chinese pines, is slightly expressed in the reproductive growth stage of the Chinese pines, and is continuously expressed at a low level, the expression quantity of the marker gene PtAP2R1 is remarkably different in the vegetative growth stage and the reproductive growth stage of the Chinese pines, and the growth and development stage of the Chinese pines can be identified according to the expression quantity of the marker gene PtAP2R1. Furthermore, by comparing the expression quantity of the marker gene PtAP2R1 in the to-be-tested Chinese pine sample with the expression quantity of the marker gene PtAP2R1 in the Chinese pine sample in the known growth stage, the growth and development stages (vegetative growth and reproductive growth) of the Chinese pine plant and even the selected branch can be efficiently and accurately identified, and guidance is provided for forestry production and forest management.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and specifically relates to marker genes PtAP2R1 Application in identifying the vegetative growth stage and reproductive growth stage of Pinus tabulaeformis. Background Art

[0002] Pinus tabuliformis Carriere is a coniferous evergreen tree belonging to the genus Pinus in the Pinaceae family. It is cold-resistant, drought-resistant, and barren-resistant, and is an important afforestation tree species in the vast areas of northern my country. After germination, Pinus tabuliformis seeds need to go through the vegetative stage and the reproductive stage, where the vegetative stage refers to the growth process of plants from seed germination, seedling growth to pre-reproduction. In forestry production practice, branches in the vegetative stage have strong regeneration ability and are more suitable for selection as asexual propagation materials for cuttings, grafting, tissue culture, etc. After trees enter reproductive growth, the advantages and disadvantages of the growth, dryness, wood and other traits of the trees will gradually appear. Therefore, the selection of excellent Pinus tabuliformis stands and germplasm resources, the determination of progeny forests, etc., mostly select trees in the reproductive growth period. However, for a long time, there have been many difficulties in determining the vegetative and reproductive growth stages of Pinus tabuliformis and the time of transition from vegetative growth to reproductive growth, and it has been impossible to identify them in vivo. Summary of the invention

[0003] The present invention aims to provide a marker gene PtAP2R1 Application of the present invention in distinguishing the vegetative growth stage and reproductive growth stage of Pinus tabulaeformis, the marker gene provided by the present invention PtAP2R1 It can be used to identify the growth and development stages of Pinus tabulaeformis plants or branches, and provide guidance for forestry production and forest management.

[0004] The present invention provides a marker gene PtAP2R1 Application of the marker gene in distinguishing the vegetative growth stage and reproductive growth stage of Pinus tabulaeformis PtAP2R1 The encoded amino acid sequence is shown in SEQ ID NO.1.

[0005] Preferably, the identification method comprises: (1) Selecting Pinus tabulaeformis tissue materials of different ages grown in the same geographical environment as the sample to be tested as a reference sample set; the Pinus tabulaeformis tissue materials in the reference sample set include Pinus tabulaeformis tissue materials in the vegetative growth stage and Pinus tabulaeformis tissue materials in the reproductive growth stage; Detecting the marker genes in the reference sample set and the sample to be tested PtAP2R1 The expression level of (2) Sort by tree age and select the marker gene in the reference sample set PtAP2R1The Pinus tabulaeformis tissue material whose expression level was significantly reduced for the first time was used as the control sample; the significantly reduced PtAP2R1 The expression level of the marker gene in the previous tree age reference sample in the reference sample set is lower than that in the previous tree age reference sample in the reference sample set. PtAP2R1 1 / 2 of the expression level; (3) Comparing the marker genes in the test sample and the control sample PtAP2R1 The expression level of the test sample is used to identify the growth and development stage of the test sample; The identification criteria are: if the marker gene in the sample to be tested PtAP2R1 The expression level of the marker gene in the control sample is lower than that in the PtAP2R1 If the expression level of the marker gene in the sample is 1 / 2, the sample to be tested is judged to be in the reproductive growth stage; PtAP2R1 The expression level of the marker gene in the control sample is equal to or greater than PtAP2R1 If the expression level is less than 1 / 2 of that of the control, the sample to be tested is judged to be in the vegetative growth stage.

[0006] Preferably, the Pinus tabulaeformis tissue material includes one or more of cambium, young shoots, branches and leaves.

[0007] Preferably, the Pinus tabulaeformis tissue material includes vascular cambium.

[0008] Preferably, the marker genes in the reference sample set and the sample to be tested are detected PtAP2R1 The method for expressing the marker gene comprises: amplifying the marker gene using an upstream primer and a downstream primer PtAP2R1 ; The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.4.

[0009] Preferably, the control sample comprises 4-year-old Pinus tabulaeformis tissue material.

[0010] Preferably, the reference sample set includes 2- to 150-year-old Pinus tabulaeformis tissue materials.

[0011] Preferably, the reference sample set includes 2- to 123-year-old Pinus tabulaeformis tissue materials.

[0012] Preferably, the Pinus tabulaeformis tissue material in the vegetative growth stage includes 2- to 6-year-old Pinus tabulaeformis tissue material; and the Pinus tabulaeformis tissue material in the reproductive growth stage includes 7- to 123-year-old Pinus tabulaeformis tissue material.

[0013] Preferably, the Pinus tabulaeformis tissue material in the vegetative growth stage includes 2- to 4-year-old Pinus tabulaeformis tissue material; and the Pinus tabulaeformis tissue material in the reproductive growth stage includes 7- to 55-year-old Pinus tabulaeformis tissue material.

[0014] The present invention provides a marker genePtAP2R1 Application of the marker gene in distinguishing the vegetative growth stage and reproductive growth stage of Pinus tabulaeformis PtAP2R1 The encoded amino acid sequence is shown in SEQ ID NO. 1. The marker gene of the present invention PtAP2R1 It is expressed in large amounts in the vegetative growth stage of Pinus tabulaeformis, in small amounts in the reproductive growth stage of Pinus tabulaeformis, and then continuously expressed at a low level. Its expression levels show extremely significant differences between the vegetative growth stage and the reproductive growth stage of Pinus tabulaeformis. PtAP2R1 The expression level of the marker gene in the pine sample to be tested can be used to identify the growth and development stage of the pine. PtAP2R1 Expression levels and marker genes in Pinus tabulaeformis samples at known growth stages PtAP2R1 The expression level of the gene can efficiently and accurately identify the growth and development stage (vegetative growth and reproductive growth) of Pinus tabulaeformis plants and even selected branches, providing guidance for forestry production and forest management. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0016] Figure 1 Gene collection for reference samples of 5-123-year-old Pinus tabulaeformis PtAP2R1 The expression level of Figure 2 Genes in the test sample and control sample PtAP2R1 The amount of expression. DETAILED DESCRIPTION

[0017] The present invention provides a marker gene PtAP2R1 Application of the marker gene in distinguishing the vegetative growth stage and reproductive growth stage of Pinus tabulaeformis PtAP2R1 The encoded amino acid sequence is shown in SEQ ID NO.1.

[0018] The marker gene of the present invention PtAP2R1The encoded amino acid sequence is shown in SEQ ID NO.1 as follows: MRTTDTSSIEPVGEITPRSSEETESACVEAATVTHIQSNNEKRRRAPFRSEMAESGTSVSSVVNSITCNENINSLEHEASSKAMELDADVISSNSTQVPPSNEGNHKIFGFSLKIETRNLISRGNLLNAIANDEQQKDPACVTRHFFPLHNGEASQVLMPTECTEGNPFIESHWTPVTSRQPENAETRKKQPENNKPVKKSRRGPRSRSSQFRGVTFYRRTGRWESHIWDCGKQVYLGGFDTAHAAARAYDRAAIKFRGVEADINFTLTDYQEDLDQTSKLSKEEFVHILRRQSTGFSRGSSKYRGVTLHKCGRWEARMGQFLGKKYIYLGLFDSEEEAARAYDKAAIKCNGKEAVTNFDPSLYEKEILEERRESQTSTGCDQNLDLSLGIGHVPTQLKKSPSYKSRAEEDSQLKRIGGSTYSNHPQDSKVLSCLTSKWGENAVPEFQVLPRGHAVYWNSQVLQVPEAMKYKESEAHRSEDNAYEQTATSGSQKYPDHLGNCKKLNMMEDLKEQSFGVPASTVSFWPMKQQQSGWTWQLHARGPTSPVFTTNKSSSPGFSPQIHTAFVPSKHGSHNMGLQWAAQSTDPCMVQTPSPHLSTRLTNGTDSSDHMVGSNLDFPGKKWKRLSVQWAARSTDPCMVQTPSPHLSTRLTNGTDSSDHMVGSNLDFPGKKIKSLSVHRDK; The marker gene PtAP2R1PtAP2R1 It is expressed in large amounts in the vegetative growth stage of Pinus tabulaeformis, in small amounts in the reproductive growth stage of Pinus tabulaeformis, and then continuously expressed at a low level. Its expression levels show extremely significant differences between the vegetative growth stage and the reproductive growth stage of Pinus tabulaeformis. PtAP2R1 The expression level of the gene can be used to identify the growth and development stage of Pinus tabulaeformis.

[0019] The identification method of the present invention preferably comprises: (1) selecting Pinus tabulaeformis tissue materials of different ages grown in the same geographical environment as the sample to be tested as a reference sample set; the Pinus tabulaeformis tissue materials in the reference sample set include Pinus tabulaeformis tissue materials in the vegetative growth stage and Pinus tabulaeformis tissue materials in the reproductive growth stage; selecting Pinus tabulaeformis tissue materials grown in the same geographical environment as the Pinus tabulaeformis tissue materials in the reference sample set as the sample to be tested; detecting the marker gene in the reference sample set and the sample to be tested PtAP2R1 The amount of expression.

[0020] The pine tissue material of the present invention preferably includes cambium, buds, branches or leaves, and more preferably vascular cambium. The reference sample set of the present invention preferably includes 2-year to 150-year-old pine tissue materials; and more preferably includes 2-year to 123-year-old pine tissue materials. The pine tissue materials in the vegetative growth stage of the present invention preferably include 2-year to 6-year-old pine tissue materials, and more preferably include 2-year to 4-year-old pine tissue materials, and more preferably 2-year-old pine tissue materials, 3-year-old pine tissue materials and 4-year-old pine tissue materials; the pine tissue materials in the reproductive growth stage preferably include 7-year to 123-year-old pine tissue materials, and more preferably include 7-55-year-old pine tissue materials, and more preferably 7-year, 13-year, 20-year, 46-year, 55-year-old pine tissue materials. Pinus tabulaeformis usually spends its juvenile period in 5 to 7 years, and forms embryos and sets seeds for the first time after 7 years. In actual production, the pine after 7 years is called the reproductive growth stage, and the pine before 7 years is called the vegetative growth stage. The marker gene of the present invention PtAP2R1 It is expressed in large quantities in Pinus tabulaeformis samples that are 2 to 4 years old and in the vegetative growth stage, and its expression level is extremely significantly reduced in Pinus tabulaeformis samples that are 7 to 123 years old and in the reproductive growth stage. It can effectively reflect and identify the growth and development stage of Pinus tabulaeformis and can be used as a molecular marker to identify the growth and development stage of Pinus tabulaeformis.

[0021] In the present invention, the marker genes in the reference sample set and the sample to be tested are detected. PtAP2R1 The method for measuring the expression level of the marker gene preferably comprises: amplifying the marker gene using an upstream primer and a downstream primer PtAP2R1; The nucleotide sequence of the upstream primer is preferably as shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is preferably as shown in SEQ ID NO.4. The present invention preferably uses Pinus tabulaeformis 18sRNA as an internal reference gene, and the nucleotide sequence of the upstream primer for amplifying the Pinus tabulaeformis internal reference gene 18sRNA is preferably as shown in SEQ ID NO.5, and the nucleotide sequence of the downstream primer is preferably as shown in SEQ ID NO.6.

[0022] (2) Sort by tree age and select the marker gene in the reference sample set PtAP2R1 The Pinus tabulaeformis tissue material in which the expression level of the marker gene was significantly reduced for the first time was used as the control sample. PtAP2R1 The expression level of the marker gene in the previous tree age reference sample in the reference sample set is lower than that in the previous tree age reference sample in the reference sample set. PtAP2R1 The transition from the vegetative growth stage to the reproductive growth stage of Pinus tabulaeformis generally occurs in 4 to 7 years. In the present invention, the control sample preferably includes a 4-year-old Pinus tabulaeformis tissue material.

[0023] (3) Comparing the marker genes in the test sample and the control sample PtAP2R1 The expression level of the marker gene in the sample to be tested is used to identify the growth and development stage of the sample to be tested; the identification standard is preferably: if the marker gene in the sample to be tested PtAP2R1 The expression level of the marker gene in the control sample is lower than that in the PtAP2R1 If the expression level of the marker gene in the sample is 1 / 2, the sample to be tested is judged to be in the reproductive growth stage; PtAP2R1 The expression level of the marker gene in the control sample is equal to or greater than PtAP2R1 If the expression level is less than 1 / 2 of that of the control, the sample to be tested is judged to be in the vegetative growth stage.

[0024] To further illustrate the present invention, the marker gene provided by the present invention is described below in conjunction with the accompanying drawings and embodiments. PtAP2R1 The application in distinguishing the vegetative growth stage and the reproductive growth stage of Pinus tabulaeformis is described in detail, but they should not be understood as limiting the scope of protection of the present invention.

[0025] Example 1 Marker genes PtAP2R1 Determination of expression level (1) Collection of reference sample sets The cambium of Pinus tabulaeformis trees aged 2, 3, 4, 7, 13, 20, 46, 55 and 123 years old were collected at the Shangzhuang Forest Farm in Xi County, Linfen City, Shanxi Province (111°10′E, 36°48′N, 1570-1930 m above sea level) at the base of the trunk, 15-20 cm above the ground, and were immediately quick-frozen with liquid nitrogen and stored in a -80°C ultra-low temperature refrigerator.

[0026] In production practice and related research, Pinus tabulaeformis undergoes a transition from vegetative growth to reproductive growth between 5 and 7 years old. The 2-year-old, 3-year-old, and 4-year-old Pinus tabulaeformis described in the present invention are in the vegetative growth stage, and the 7-year-old, 13-year-old, 20-year-old, 46-year-old, 55-year-old, and 123-year-old Pinus tabulaeformis are in the reproductive growth stage.

[0027] (2) Extraction of total RNA from reference sample set The total RNA of the standard samples collected above was extracted using RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) from Tiangen Biochemical Technology (Beijing) Co., Ltd. The RNA concentration and integrity were determined by Nano micro-spectrophotometer and 1% agarose gel electrophoresis, respectively.

[0028] (3) Reverse transcription of total RNA from reference sample set In an RNase free PCR tube, complete the reverse transcription reaction of the total RNA obtained in step (2) and remove the genomic DNA. Prepare the reverse transcription system according to Table 1: Table 1 Reverse transcription system

[0029] After gently mixing the reaction system in Table 1, incubate at 42°C for 30 min; heat at 85°C for 5 s to inactivate TransScriptRT / RI and gDNA Remover, and store the cDNA at -20°C.

[0030] (4) Primer design The NCBI primer online design website Primer-BLAST and NCBI Conserved Domain Search were used to design the internal reference gene 18sRNA and gene of Pinus tabulaeformis. PtAP2R1 (SEQ ID NO.2) primers, gene 18sRNA and gene PtAP2R1 The primer sequences are as follows: PtAP2R1-F (SEQ ID NO.3): 5'-CCTCTTCATAACGGGGAGGC-3'; PtAP2R1-R (SEQ ID NO.4): 5'-GCATTCTCTGGTTGACGGGA-3'; 18sRNA-F (SEQ ID NO.5): 5′-CGGCTACCACATCCAAGGAA-3′; 18sRNA-R (SEQ ID NO. 6): 5′-GCTGGAATTACCGCGGCT-3′.

[0031] (5) Genes PtAP2R1 Real-time fluorescence quantitative PCR (qRT-PCR) detection experiment The cDNA of the reverse transcription product in step (3) was diluted 50 times and used as a template to detect the gene according to the fluorescence quantitative PCR amplification system in Table 2 below. PtAP2R1 The expression level in the standard sample collected in step (1), the reagents and amounts used in the amplification system were referred to the instructions of the TransScript One-Step gDAN Removal and cDNA Synthesis SuperMix TransScript Kit of Quanshijin Company. Each sample was repeated three times in technical replicates and three times in biological replicates. 18sRNA was used as the internal reference in each plate of samples.

[0032] Table 2 qRT-PCR reaction system

[0033] The SYBR Green chimeric fluorescence method was used for the reaction. The pre-denaturation reaction program was set at 95°C for 2 min; the PCR reaction program was set at 94°C for 5 s, 60°C for 30 s, and 39 cycles; the termination extension reaction program was set at 94°C for 5 s; and the melting curve analysis reaction program was set at 65~95°C with an increase of 0.5°C every 5 s.

[0034] Gene PtAP2R1 The expression levels in the reference sample set are as follows Figure 1 Shown: Gene PtAP2R1 It is highly expressed in 2-year-old and 3-year-old Pinus tabulaeformis, and the gene in 2-year-old Pinus tabulaeformis PtAP2R1 The expression level of the gene in 1- and 3-year-old pines is recorded as PtAP2R1 The expression level of gene PtAP2R1 The expression level in 4-year-old Pinus tabulaeformis decreased significantly to 0.34; it was slightly expressed in 7-123-year-old Pinus tabulaeformis, with the expression level not exceeding 0.1. PtAP2R1 The expression levels differ significantly during the transition from vegetative growth to reproductive growth, which can effectively reflect and identify the growth and development stage of Pinus tabulaeformis.

[0035] Example 2 (1) Selection of control samples and setting of identification standards The results of step (5) of Example 1 reflect that the marker gene PtAP2R1 The expression levels of 4-year-old Pinus tabulaeformis samples in the vegetative growth stage were significantly different, so the samples were selected as control samples for judging the growth and development stage of Pinus tabulaeformis. PtAP2R1 The expression level of the marker gene in the control sample is lower than that in the PtAP2R1If the expression level of the marker gene in the sample is 1 / 2, the sample to be tested is judged to be in the reproductive growth stage; PtAP2R1 The expression level of the marker gene in the control sample is equal to or greater than PtAP2R1 If the expression level is less than 1 / 2 of that of the control, the sample to be tested is judged to be in the vegetative growth stage.

[0036] (2) Collection of test samples and control samples Branches from improved varieties of Pinus tabulaeformis were randomly collected, and the vascular cambium was scraped off as the test samples; at the same time, 4-year-old Pinus tabulaeformis with known age in the same geographical location and growth environment was selected, and the cambium at the base of the trunk 15-20 cm above the ground was taken as the control sample. The above materials were immediately frozen with liquid nitrogen after collection and stored in a -80℃ ultra-low temperature refrigerator.

[0037] (3) Extraction of total RNA from test samples and control samples The total RNA extraction of the test samples and the control samples was the same as that in step (2) of Example 1.

[0038] (4) Reverse transcription of total RNA from test and control samples The reverse transcription of total RNA of the test sample and the control sample is the same as the method in step (3) of Example 1.

[0039] (5) PtAP2R1 Real-time fluorescence quantitative PCR (qRT-PCR) detection of genes PtAP2R1 The real-time fluorescence quantitative PCR (qRT-PCR) detection of the gene was the same as the method in step (5) of Example 1. The results are as follows Figure 2 As shown, the genes in the control sample PtAP2R1 The expression level of the gene in the sample to be tested is recorded as 1. PtAP2R1 The expression level of the gene in the sample to be tested is 0.31. PtAP2R1 The expression level of the tested sample was lower than 1 / 2 of that of the control sample. Therefore, the developmental stage of the tested sample was judged to be the reproductive growth stage.

[0040] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Marker genes PtAP2R1 Application of the marker gene in distinguishing the vegetative growth stage and reproductive growth stage of Pinus tabulaeformis PtAP2R1 The encoded amino acid sequence is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The identification method comprises: (1) Selecting Pinus tabulaeformis tissue materials of different ages grown in the same geographical environment as the sample to be tested as a reference sample set; the Pinus tabulaeformis tissue materials in the reference sample set include Pinus tabulaeformis tissue materials in the vegetative growth stage and Pinus tabulaeformis tissue materials in the reproductive growth stage; Detecting the marker genes in the reference sample set and the sample to be tested PtAP2R1 The expression level of (2) Sort by tree age and select the marker gene in the reference sample set PtAP2R1 The Pinus tabulaeformis tissue material whose expression level was significantly reduced for the first time was used as the control sample; the significantly reduced PtAP2R1 The expression level of the marker gene in the previous tree age reference sample in the reference sample set is lower than that in the previous tree age reference sample in the reference sample set. PtAP2R1 1 / 2 of the expression level; (3) Comparing the marker genes in the test sample and the control sample PtAP2R1 The expression level of the test sample is used to identify the growth and development stage of the test sample; The identification criteria are: if the marker gene in the sample to be tested PtAP2R1 The expression level of the marker gene in the control sample is lower than that in the PtAP2R1 If the expression level of the marker gene in the sample is 1 / 2, the sample to be tested is judged to be in the reproductive growth stage; PtAP2R1 The expression level of the marker gene in the control sample is equal to or greater than PtAP2R1 If the expression level is less than 1 / 2 of that of the control, the sample to be tested is judged to be in the vegetative growth stage.

3. The use according to claim 2, characterized in that: The Pinus tabulaeformis tissue material comprises one or more of cambium, young shoots, branches and leaves.

4. The use according to claim 3, characterized in that: The Chinese pine tissue material comprises a vascular cambium.

5. The use according to claim 2, characterized in that: Detecting the marker genes in the reference sample set and the sample to be tested PtAP2R1 The method for expressing the marker gene comprises: amplifying the marker gene using an upstream primer and a downstream primer PtAP2R1 ; The nucleotide sequence of the upstream primer is shown in SEQ ID NO.3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO.

4.

6. The use according to claim 2, characterized in that: The control samples include 4-year-old Pinus tabulaeformis tissue materials.

7. The use according to claim 2, characterized in that: The reference sample set includes 2- to 150-year-old Pinus tabulaeformis tissue materials.

8. The use according to claim 7, characterized in that: The reference sample set includes 2- to 123-year-old Pinus tabulaeformis tissue materials.

9. The use according to claim 8, characterized in that: The pine tissue materials in the vegetative growth stage include 2- to 6-year-old pine tissue materials; the pine tissue materials in the reproductive growth stage include 7- to 123-year-old pine tissue materials.

10. The use according to claim 9, characterized in that: The pine tissue material in the vegetative growth stage includes 2- to 4-year-old pine tissue material; the pine tissue material in the reproductive growth stage includes 7- to 55-year-old pine tissue material.

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