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CN-122012547-A - Application of PmACAA gene in judging activity of masson pine seeds

CN122012547ACN 122012547 ACN122012547 ACN 122012547ACN-122012547-A

Abstract

The invention relates to the technical field of plant detection, in particular to an application of PmACAA gene in judging the vigor of Pinus massoniana seeds. According to the invention, the activity of the masson pine seeds is judged by testing the relative expression quantity of PmACAA genes, and the expression quantity of acetyl-CoA acyltransferase is regulated downwards, so that the activity of the masson pine seeds is high. The invention can realize high-efficiency and rapid determination of the vigor of the masson pine seeds through PmACAA genes, and is not limited by the germination period of the seeds. The method provided by the invention greatly improves the efficiency of the activity test of the masson pine seeds, provides a good early tool for the storage, the later cultivation and the like of the masson pine seeds, and has good economic and social benefits.

Inventors

  • WANG HAOYUN
  • WU FENG
  • LEI BIN
  • You Sujie
  • TAO GUANGXU
  • LU DAN

Assignees

  • 贵州大学

Dates

Publication Date
20260512
Application Date
20260302

Claims (10)

  1. 1. The application of PmACAA gene in judging the vigor of masson pine seed is characterized in that the base sequence of PmACAA gene is shown as SEQ ID NO. 1.
  2. 2. The use of the PmACAA protein coded by PmACAA gene in determining the viability of masson pine seeds according to claim 1, wherein the amino acid sequence of PmACAA protein is shown as SEQ ID NO. 2.
  3. 3. A primer pair for amplifying PmACAA genes according to the above scheme.
  4. 4. The primer pair of claim 3, wherein the primer pair comprises a forward primer and a reverse primer, the forward primer and the reverse primer having nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4, respectively.
  5. 5. A kit comprising the primer pair and PCR amplification reagents of the above scheme.
  6. 6. The kit of claim 5, further comprising a cDNA template and rnase free water.
  7. 7. The kit according to claim 5, wherein the kit further comprises primers for internal reference genes, wherein the internal reference genes are 18s rRNA gene and UBC gene.
  8. 8. Use of the primer pair of any one of claims 3 to 4 or the kit of any one of claims 5 to 7 for determining the vigor of a pinus massoniana seed.
  9. 9. A method for determining the viability of a masson pine seed comprising the steps of: taking cDNA of a sample to be detected and cDNA of a reference sample as templates, respectively carrying out fluorescent quantitative PCR amplification by using the primer pair according to the scheme, and respectively counting the expression quantity of PmACAA genes in the sample to be detected and the reference sample; when the expression quantity of PmACAA gene in the sample to be detected is lower than that of PmACAA gene in the reference sample, judging that the activity of the sample to be detected is higher than that of the reference sample.
  10. 10. The method of claim 9, wherein the reaction system for fluorescent quantitative PCR amplification is 10 μl, comprising: 5. mu.L of 2X SuperReal PreMix Plus, 1. Mu.L of cDNA template, 0.5. Mu.L of 100. Mu.M upstream primer, 0.5. Mu.L of 100. Mu.M downstream primer and 3. Mu.L of RNase-free water.

Description

Application of PmACAA gene in judging activity of masson pine seeds Technical Field The invention relates to the technical field of plant detection, in particular to application of PmACAA (acetyl-CoA acyltransferase I) gene in judging the vigor of pinus massoniana seeds. Background Pinus massoniana is a arbor plant of Pinaceae Pinus, the flowering period is 4-5 months, and cones are ripe in 10-12 months of the next year. It is widely distributed in the eastern wet area of subtropical zone in China, and is the main afforestation tree species in south China. The pinus massoniana is suitable for barren mountain forestation and ecological landscape forestation, and also can be used for roadside and garden greening, and the wood can be used for paper making, building and furniture manufacturing, and has important ecological value and economic value. The seeds are core carriers for storing the wood germplasm resources, the pinus massoniana is used as the main forestation pioneer tree seeds and wood strategic reserve resources in the south of China, the vitality of the seeds is more than 90 percent under normal conditions, but the obvious phenomenon of the plump lean year exists in the tree seeds, the vitality of the seeds is reduced in the long-term storage or stress exposure process, and the problems of low emergence rate, uneven seed germination, poor seedling uniformity, weak seedling stress resistance, low forestation survival rate and the like are caused. Based on this, technicians often determine their viability by using embryo transformation methods, and measure their activity by observing the color change (yellow, green, white and hard) of embryo, but TTC staining methods rely on subjective judgment of the shade of staining (e.g., dark red, light red), lack of clear quantitative criteria, and result in poor consistency of interpretation of results and lower detection efficiency. Disclosure of Invention In view of the above, the invention provides an application of PmACAA gene in judging the vigor of the masson pine seeds, and the invention can efficiently and rapidly judge the vigor of the masson pine seeds through PmACAA gene without being limited by the germination period of the seeds. The invention provides an application of PmACAA gene in judging the vigor of masson pine seeds, wherein the base sequence of PmACAA gene is shown as SEQ ID NO. 1. The invention also provides application of the PmACAA gene coded PmACAA protein in judging the vigor of the masson pine seeds, wherein the amino acid sequence of the PmACAA protein is shown as SEQ ID NO. 2. The invention also provides a primer pair for amplifying the PmACAA gene in the scheme. Preferably, the primer pair comprises a forward primer and a reverse primer, and the nucleotide sequences of the forward primer and the reverse primer are respectively shown as SEQ ID NO.3 and SEQ ID NO. 4. The invention also provides a kit which comprises the primer pair and the PCR amplification reagent according to the scheme. Preferably, the kit further comprises a cDNA template and RNase-free water. Preferably, the kit further comprises primers of internal reference genes, wherein the internal reference genes are 18s rRNA genes and UBC genes. The invention also provides application of the primer pair or the kit in the scheme in judging the vigor of the masson pine seeds. The invention also provides a method for judging the vigor of the pinus massoniana seeds, which comprises the following steps of taking cDNA of a sample to be detected and cDNA of a reference sample as templates, respectively carrying out fluorescent quantitative PCR amplification by using the primer pair according to the scheme, respectively counting the expression quantity of PmACAA genes in the sample to be detected and the reference sample, and judging that the vigor of the sample to be detected is higher than that of the reference sample when the expression quantity of PmACAA genes in the sample to be detected is lower than that of PmACAA genes in the reference sample. Preferably, the reaction system for fluorescent quantitative PCR amplification comprises, in 10. Mu.L, 5. Mu.L of 2X SuperReal PreMix Plus, 1. Mu.L of cDNA template, 0.5. Mu.L of 100. Mu.M upstream primer, 0.5. Mu.L of 100. Mu.M downstream primer and 3. Mu.L of RNase-free water. The invention has the following beneficial effects: According to the invention, the activity of the masson pine seeds is judged by testing the relative expression quantity of PmACAA genes, and the expression quantity of acetyl-CoA acyltransferase is regulated downwards, so that the activity of the masson pine seeds is high. The invention can realize high-efficiency and rapid determination of the vigor of the masson pine seeds through PmACAA genes, and is not limited by the germination period of the seeds. The method provided by the invention greatly improves the efficiency of the activity test of the masson pine seeds, provides a good early-stage tool for c