CN102876685A - Cloning and application of PcBLFY (Pistacia chinensis Bunge blooming gene) - Google Patents
Cloning and application of PcBLFY (Pistacia chinensis Bunge blooming gene) Download PDFInfo
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- 230000009261 transgenic effect Effects 0.000 abstract 1
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Abstract
本发明公开了一种黄连木开花基因PcBLFY，本发明还公开了一种黄连木开花基因的克隆，根据其它植物已知LEAFY基因序列，设计兼并引物，以黄连木雄株、雌株和雌雄同株总RNA为模板，采用RT-PCR方法合成cDNA第一条链，5’RACE和3’RACE方法克隆PcBLFY的cDNA全长序列。根据获得的cDNA序列设计引物，通过RT-PCR的方法从黄连木雄株、雌株、雌雄同株植株中分别获得2个、1个、2个序列存在单碱基位点多态性差异的LEAFY基因。本发明公开的内容可供创造早开花结果的转基因育种使用。本发明的重要应用前景是用于黄连木不同性别植株的苗期性别鉴定，用于调节黄连木能源林的植株性别比例，从而大幅提高产量。
The invention discloses a Pistacia flowering gene PcBLFY . The invention also discloses a clone of the Pistacia flowering gene. According to the known LEAFY gene sequence of other plants, an amalgamative primer is designed to use the male, female and hermaphroditic plants of Pistacia. The total RNA of the strain was used as a template, the first strand of cDNA was synthesized by RT-PCR method, and the full-length cDNA sequence of PcBLFY was cloned by 5'RACE and 3'RACE methods. Primers were designed according to the obtained cDNA sequence, and 2, 1, and 2 sequences with single-base polymorphism differences were obtained from male, female, and monoecious plants of Pistacia chinensis by RT-PCR. The LEAFY gene. The present disclosure can be used in transgenic breeding for the creation of early flowering and fruiting. The important application prospect of the present invention is that it can be used for sex identification of plants of different sexes in the seedling stage of Pistacia chinensis, and can be used for adjusting the sex ratio of plants in Pistacia chinensis energy forests, so as to greatly increase the yield.
Description
技术领域 technical field
本发明涉及生物技术与现代农业技术领域，具体涉及一种黄连木开花控制基因（PcBLFY）的克隆及其应用。The invention relates to the fields of biotechnology and modern agricultural technology, in particular to the cloning and application of a pistachio flowering control gene (PcBLFY).
背景技术 Background technique
化石能源日趋枯竭，加之化石类能源使用过程中产生的有毒气体和温室气体带来的环境影响，开发绿色可再生的清洁能源成为世界各国争相研究的领域。其中生物能源是最有前景的发展方向之一。我国是能源消费大国，60%的石油依赖进口，发展生物柴油替代有限的石油资源意义重大。With the depletion of fossil energy, coupled with the environmental impact of toxic gases and greenhouse gases produced during the use of fossil energy, the development of green, renewable and clean energy has become a field of research that countries around the world are vying for. Among them, bioenergy is one of the most promising development directions. my country is a big energy consumer, and 60% of its oil depends on imports. It is of great significance to develop biodiesel to replace limited oil resources.
目前，生物柴油产业的瓶颈问题是原料的供应问题，相关生物柴油生产企业普遍开工率低，只有10%左右；其主要原因是原料油脂供应不足，且油脂成本占企业生产成本比例高，达60-70%。我国人多地少，不能象发达国家一样走利用耕地实现生物柴油原料油供应的路子，利用荒山、荒地等边际性土地种植油料植物实现原料油脂的供应是我国生物柴油产业发展的必由之路。At present, the bottleneck problem of the biodiesel industry is the supply of raw materials. The operating rate of relevant biodiesel production enterprises is generally low, only about 10%. -70%. Our country has a large population and little land, so we cannot take the path of using cultivated land to supply biodiesel raw material oil like developed countries. Using barren hills, barren land and other marginal lands to plant oil plants to realize the supply of raw material oil is the only way for the development of my country's biodiesel industry.
黄连木(Pistacia chinensis Bunge)是一种重要的多年生木本油料植物，分布范围广，可在荒山荒地等边际性土地上种植，适宜大面积造林，而且种子含油率高达40%以上。黄连木种子油脂是生物柴油的优质原料，黄连木被认为是未来生态能源林建设和生物质柴油产业优先发展树种。由于黄连木发展前景看好，目前针对黄连木的研究越来越多，但基本集中在资源调查、育苗、防治、栽培技术等宏观方面的研究，有关遗传基础、丰产技术等方面的研究较少。Pistacia chinensis Bunge is an important perennial woody oil plant with a wide range of distribution. It can be planted on marginal lands such as barren mountains and wastelands. It is suitable for large-scale afforestation, and the oil content of its seeds is as high as 40%. Pistacia chinensis seed oil is a high-quality raw material for biodiesel, and Pistacia chinensis is considered to be a priority tree species for future ecological energy forest construction and biodiesel industry. Due to the promising development prospects of Pistacia chinensis, there are more and more researches on Pistacia chinensis, but they basically focus on macroscopic aspects such as resource investigation, seedling cultivation, control, and cultivation techniques, while there are few studies on genetic basis and high-yield techniques.
黄连木为雌雄异株植物，天然次生林中的雌雄株的比例大约为1:1，而10%左右的雄株即可以保证充足的花粉供应，因而天然次生林中雄株过剩，能结果的雌株比例偏低（在天然次生林中偶见有雌雄同株的个体，目前还无法在生产上应用）。而黄连木童期长，8-12年才能开花结果，在开花结果前没有有效的雌雄株的鉴定方法。因而对黄连木开花主控基因的克隆和功能研究，以及稳定实用的黄连木苗期雌雄鉴定技术的研究，对于研发童期短的早结果株系、培育雌株比例高的高产黄连木能源林，从而大幅提高黄连木的单位面积产量具有重要意义。Pistacia chinensis is a dioecious plant. The ratio of male and female plants in natural secondary forests is about 1:1, and about 10% of male plants can ensure sufficient pollen supply. Therefore, there are excess male plants in natural secondary forests, and the female plants that can bear fruit The ratio is low (monoecious individuals are occasionally seen in natural secondary forests, which cannot be used in production at present). However, Pistacia chinensis has a long childhood period, and it takes 8-12 years to bloom and bear fruit, and there is no effective identification method for male and female plants before flowering and bear fruit. Therefore, the cloning and functional research of the main gene controlling flowering of Pistacia chinensis, as well as the research on the stable and practical identification technology of the male and female of Pistacia chinensis seedlings, are of great importance for the development of early-bearing strains with short childhood and high-yielding Pistacia chinensis energy forests with a high proportion of female plants. , It is of great significance to greatly increase the yield per unit area of Pistacia chinensis.
发明内容 Contents of the invention
本发明的目的是用于黄连木不同性别植株的苗期性别鉴定，用于调节黄连木能源林的植株性别比例，从而大幅提高产量。The purpose of the present invention is to be used for the gender identification of plants of different sexes of Pistacia chinensis at the seedling stage, and to adjust the sex ratio of the plants of Pistacia chinensis energy forest, thereby greatly increasing the yield.
为了实现上述目的本发明采用如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
根据其它植物已知LEAFY基因序列，设计兼并引物，以黄连木雄株、雌株和雌雄同株总RNA为模板，采用RT-PCR 方法合成cDNA第一条链，5’RACE和3’RACE方法克隆PcBLFY的cDNA全长序列。根据获得的cDNA序列设计引物，通过RT-PCR的方法从黄连木雄株、雌株、雌雄同株植株中分别获得2个、1个、2个序列存在单碱基位点多态性差异的LEAFY基因。According to the known LEAFY gene sequences of other plants, design degenerate primers, use the total RNA of Pistacia chinensis male, female and monoecious plants as templates, and use RT-PCR to synthesize the first strand of cDNA, 5'RACE and 3'RACE methods The full-length cDNA sequence of PcBLFY was cloned. Primers were designed according to the obtained cDNA sequence, and 2, 1, and 2 sequences with single-base polymorphism differences were obtained from male, female, and monoecious plants of Pistacia chinensis by RT-PCR. The LEAFY gene.
一种黄连木开花基因（PcBLFY）具有如下cDNA核苷酸序列：A Pistacia flowering gene (PcBLFY) has the following cDNA nucleotide sequence:
0001 CAAGTGTCAAGTTTCAAACAAACAAAACATCATCAACTCCCCTTTCACTT0001 CAAGTGTCAAGTTTCAAACAAACAAAACATCATCAACTCCCCTTTCACTT
0051 CTTTAGTTACCCAATCATAAAACAATTAAAACCAGTGACTTAAAGGGCAG0051 CTTTAGTTACCCAATCATAAACAATTAAAACCAGTGACTTAAAGGGCAG
0101 TTTTGTGAACCCAAAAACAGTTTGCCTTTATAACAAAGCAACACCAACAC0101 TTTTGTGAACCCAAAAACAGTTTGCCTTTATAACAAAGCAACACCAACAC
0151 ACAGCTCAAAATYTCAGTTGCTCAGAGAGATGGATCCTGAAGCATTCACG0151 ACAGCTCAAAATYTCAGTTGCTCAGAGAGATGGATCCTGAAGCATTCACG
0201 GCGAGCTTGTTTAAGTGGGACCCGCGTGGGSTGGTGCCGCCACAGACCAG0201 GCGAGCTTGTTTAAGTGGGACCCGCGTGGGSTGGTGCCGCCACAGACCAG
0251 GGTGCTGGAACCGGTGGTGCCACTACCGGCAGCGATTTCCGCGGCCACCG0251 GGTGCTGGAACCGGTGGTGCCACTACCGGCAGCGATTTCCGCGGCCACCG
0301 CCTTCTCGGTGGTGCGTCCAAGGGAGCTAGGTGGGCTGGAGGAGTTGTTC0301 CCTTCTCGGTGGTGCGTCCAAGGGAGCTAGGTGGGCTGGAGGAGTTGTTC
0351 CAGGCTTATGGGATAAGGTACTACACGGCAGCGAAGATAGCGGAGCTTGG0351 CAGGCTTATGGGATAAGGTACTACACGGCAGCGAAGATAGCGGAGCTTGG
0401 GTTCACGGTGAATACACTTGTGAATATGAAAGATGAGGAGCTTGATGAGA0401 GTTCACGGTGAATACACTTGTGAATATGAAAGATGAGGAGCTTGATGAGA
0451 TGATGAACAGCCTGTCTCAGATATTTAGGTGGGAACTCCTTGTTGGAGAG0451 TGATGAACAGCCTGTCTCAGATATTTAGGTGGGAACTCCTTGTTGGAGAG
0501 AGGTATGGTATTAAAGCTGCTGTTAGAGCTGAGAGGAGAAGGCTTGATGA0501 AGGTATGGTATTAAAGCTGCTGTTAGAGCTGAGAGGAGAAGGCTTGATGA
0551 GGAGGATTCACGAAGGCGTCACWTACTGTCTGGTGATACTACCAACTCTG0551 GGAGGATTCACGAAGGCGTCACWTACTGTCTGGTGATACTACCAACTCTG
0601 TTGATGCTTTGTCCCAAGAAGGGTTATCTGAGGAACCAGTGCAGCAAGAG0601 TTGATGCTTTGTCCCAAGAAGGGTTATCTGAGGAACCAGTGCAGCAAGAG
0651 AAGGAGGCAGCAGGGAGCGGAGGAGGAGGAACGTGGGACTTAGCGGTGGC0651 AAGGAGGCAGCAGGGAGCGGAGGAGGAGGAACGTGGGACTTAGCGGTGGC
0701 AGTGACGGAGAGGAAGAAACAGCGGCGGAGGAAGGGGCAAAGGAAGATAG0701 AGTGACGGAGAGGAAGAAACAGCGGCGGAGGAAGGGGCAAAGGAAGATAG
0751 TGGATGTTAATCACTATGATGAAAATGAAGAYGATGAGAATGCTGAAGGC0751 TGGATGTTAATCACTATGATGAAAATGAAGAYGATGAGAATGCTGAAGGC
0801 AGTGAGAGACAACGAGAGCACCCCTTCATAGTGACGGAGCCTGGTGAAGT0801 AGTGAGAGACAACGAGAGCACCCCTTCATAGTGACGGAGCCTGGTGAAGT
0851 GGCACGTGGCAAAAAAAACGGCCTTGATTACCTGTTCCATCTCTATGAGC0851 GGCACGTGGCAAAAAAAACGGCCTTGATTACCTGTTCCATCTCTATGAGC
0901 AATGCCGTGATTTCTTGATCCAGGTCCAGAACATCGCCAAGGAGCGTGGC0901 AATGCCGTGATTTCTTGATCCAGGTCCAGAACATCGCCAAGGAGCGTGGC
0951 GAAAAATGCCCAACCAAGGTGACAAATCAGGTGTTTAGGTATGCAAAGAA0951 GAAAAATGCCCAACCAAGGTGACAAATCAGGTGTTTAGGTATGCAAAGAA
1001 GGCTGGTGCAAGCTACATTAACAAGCCAAAGATGAGACACTATGTGCACT1001 GGCTGGTGCAAGCTACATTAACAAGCCAAAGATGAGACACTATGTGCACT
1051 GCTATGCGTTGCACTGCCTGGACGAGGAGGCATCAGATGCACTGAGGAGG1051 GCTATGCGTTGCACTGCCTGGACGAGGAGGCATCAGATGCACTGAGGAGG
1101 GTTTTCAAGGAAAGAGGAGAGAACGTGGGGGCTTGGAGACAGGCTTGTTA1101 GTTTTCAAGGAAAGAGGAGAGAACGTGGGGGCTTGGAGACAGGCTTGTTA
1151 TAAGCCACTTGTGGGCATTGCAGCACGGCAAGGCTGGGATATTGATGCCA1151 TAAGCCACTTGTGGGCATTGCAGCACGGCAAGGCTGGGATATTGATGCCA
1201 TTTTCAATGCGCATCCTCGTCTGGCCATTTGGTATGTGCCCACGAAGCTG1201 TTTTCAATGCGCATCCTCGTCTGGCCATTTGGTATGTGCCCACGAAGCTG
1251 CGTCAACTTTGTCATGCTGAGCGCAATAGTGTCRCTGCTTCTAGCTCTGT1251 CGTCAACTTTGTCATGCTGAGCGCAATAGTGTCRCTGCTTCTAGCTCTGT
1301 GTCTGGCGGGGCTGATCAGCTGCCTTTCTAAATTAAAGCCTCACTGAATG1301 GTCTGGCGGGGCTGATCAGCTGCCTTTCTAAATTAAAGCCTCACTGAATG
1351 ACCGATTTGCCTAGTGATTAATCCTATTTATGGTGTCTGTTAGCAGTGTT1351 ACCGATTTGCCTAGTGATTAATCCTATTTATGGTGTCTGTTAGCAGTGTT
1401 TATTCGTGAATGTTTGTAATCTCACTTCATTTAGACTTATAGCAACTAAT1401 TATTCGTGAATGTTTGTAATCTCACTTCATTTAGACTTATAGCAACTAAT
1451 TAATCACARTATTATCACATCAATCGTACGTGTTCTTTAATATTTCTTTT1451 TAATCACARTATTATCACACAATCGTACGTGTTCTTTAATATTTCTTTT
1501 ATCTTATTTGATTTATTATCCATAAAACATTTCCGTAGAAAAAAAAAAAA1501 ATCTTATTTGATTTATTATCCATAAAACATTTCCGTAGAAAAAAAAAAAAA
1551 AAAAAAAAAAAAAAAAAAAA1551 AAAAAAAAAAAAAAAAAAAAA
其中，R：A 或G； Y:C或T； S:G或C； W:A或T(以黑体显示)Among them, R: A or G; Y: C or T; S: G or C; W: A or T (shown in bold)
黄连木开花基因的制备方法包括如下步骤：（a）提取黄连木雌雄株和两性株总RNA，并以其为模板，通过RT-PCR方法合成CDNA第一链；（b）采用5’RACE和3’RACE的方法克隆获得全长cDNA。The preparation method of the flowering gene of Pistacia chinensis comprises the following steps: (a) extracting the total RNA of male and female plants and hermaphrodites of Pistacia chinensis, and using it as a template to synthesize the first strand of cDNA by RT-PCR; (b) using 5' RACE and The full-length cDNA was cloned by 3'RACE method.
分别提取黄连木雄株、雌株、雌雄同株植物总RNA，扩增三种类型植株编码区序列；（a）黄连木雄株中获得两种LEAFY基因（PcBLFYM1、PcBLFYM2）；（b）雌株中获得一种LEAFY基因（PcBLFYF）；（c）雌雄同株植物中获得两种LEAFY基因（PcBLFYF, PcBLFYM3）。The total RNA of male, female and monoecious plants of Pistacia chinensis were extracted respectively, and the coding region sequences of three types of plants were amplified; (a) two LEAFY genes (PcBLFYM1, PcBLFYM2) were obtained from male plants of Pistacia chinensis; (b) One LEAFY gene (PcBLFYF) was acquired in one plant; (c) Two LEAFY genes (PcBLFYF, PcBLFYM3) were acquired in monoecious plants.
上述LEAFY基因的核苷酸序列为:The nucleotide sequence of the above-mentioned LEAFY gene is:
PcBLFYM1：PcBLFYM1:
0001 ATGGATCCTGAAGCATTCACGGCGAGCTTGTTTAAGTGGGACCCGCGTGG0001 ATGGATCCTGAAGCATTCACGGCGAGCTTGTTTAAGTGGGACCCGCGTGG
0051 GGTGGTGCCGCCACAGACCAGGGTGCTGGAACCGGTGGTGCCACTACCGG0051 GGTGGTGCCGCCACAGACCAGGGTGCTGGAACCGGTGGTGCCACTACCGG
0101 CAGCGATTTCCGCGGCCACCGCCTTCTCGGTGGTGCGTCCAAGGGAGCTA0101 CAGCGATTTCCGCGGCCACCGCCTTCTCGGTGGTGCGTCCAAGGGAGCTA
0151 GGTGGGCTGGAGGAGTTGTTCCAGGCTTATGGGATAAGGTACTACACGGC0151 GGTGGGCTGGAGGAGTTGTTCCAGGCTTATGGGATAAGGTACTACACGGC
0201 AGCGAAGATAGCGGAGCTTGGGTTCACGGTGAATACACTTGTGAATATGA0201 AGCGAAGATAGCGGAGCTTGGGTTCACGGTGAATACACTTGTGAATATGA
0251 AAGATGAGGAGCTTGATGAGATGATGAACAGCCTGTCTCAGATATTTAGG0251 AAGATGAGGAGCTTGATGAGATGATGAACAGCCTGTCTCAGATATTTAGG
0301 TGGGAACTCCTTGTTGGAGAGAGGTATGGTATTAAAGCTGCTGTTAGAGC0301 TGGGAACTCCTTGTTGGAGAGAGGTATGGTATTAAAGCTGCTGTTAGAGC
0351 TGAGAGGAGAAGGCTTGATGAGGAGGATTCACGAAGGCGTCACATACTGT0351 TGAGAGGAGAAGGCTTGATGAGGAGGATTCACGAAGGCGTCACATACTGT
0401 CTGGTGATACTACCAACTCTGTTGATGCTTTGTCCCAAGAAGGGTTATCT0401 CTGGTGATACTACCAACTCTGTTGATGCTTTGTCCCAAGAAGGGTTATCT
0451 GAGGAACCAGTGCAGCAAGAGAAGGAGGCAGCAGGGAGCGGAGGAGGAGG0451 GAGGAACCAGTGCAGCAAGAGAAGGAGGCAGCAGGGAGCGGAGGAGGAGG
0501 AACGTGGGACTTAGCGGTGGCAGTGACGGAGAGGAAGAAACAGCGGCGGA0501 AACGTGGGACTTAGCGGTGGCAGTGACGGAGAGGAAGAAACAGCGGCGGA
0551 GGAAGGGGCAAAGGAAGATAGTGGATGTTAATCACTATGATGAAAATGAA0551 GGAAGGGGCAAAGGAAGATAGTGGATGTTAATCACTATGATGAAAATGAA
0601 GACGATGAGAATGCTGAAGGCAGTGAGAGACAACGAGAGCACCCCTTCAT0601 GACGATGAGAATGCTGAAGGCAGTGAGAGACAACGAGAGCACCCCTTCAT
0651 AGTGACGGAGCCTGGTGAAGTGGCACGTGGCAAAAAAAACGGCCTTGATT0651 AGTGACGGAGCCTGGTGAAGTGGCACGTGGCAAAAAAAACGGCCTTGATT
0701 ACCTGTTCCATCTCTATGAGCAATGCCGTGATTTCTTGATCCAGGTCCAG0701 ACCTGTTCCATCTCTATGAGCAATGCCGTGATTTCTTGATCCAGGTCCAG
0751 AACATCGCCAAGGAGCGTGGCGAAAAATGCCCAACCAAGGTGACAAATCA0751 AACATCGCCAAGGAGCGTGGCGAAAAATGCCCAACCAAGGTGACAAATCA
0801 GGTGTTTAGGTATGCAAAGAAGGCTGGTGCAAGCTACATTAACAAGCCAA0801 GGTGTTTAGGTATGCAAAGAAGGCTGGTGCAAGCTACATTAACAAGCCAA
0851 AGATGAGACACTATGTGCACTGCTATGCGTTGCACTGCCTGGACGAGGAG0851 AGATGAGACACTATGTGCACTGCTATGCGTTGCACTGCCTGGACGAGGAG
0901 GCATCAGATGCACTGAGGAGGGTTTTCAAGGAAAGAGGAGAGAACGTGGG0901 GCATCAGATGCACTGAGGAGGGTTTTCAAGGAAAGAGGAGAGAACGTGGG
0951 GGCTTGGAGACAGGCTTGTTATAAGCCACTTGTGGGCATTGCAGCACGGC0951 GGCTTGGAGACAGGCTTGTTATAAGCCACTTGTGGGCATTGCAGCACGGC
1001 AAGGCTGGGATATTGATGCCATTTTCAATGCGCATCCTCGTCTGGCCATT1001 AAGGCTGGGATATTGATGCCATTTTCAATGCGCATCCTCGTCTGGCCATT
1051 TGGTATGTGCCCACGAAGCTGCGTCAACTTTGTCATGCTGAGCGCAATAG1051 TGGTATGTGCCCACGAAGCTGCGTCAACTTTGTCATGCTGAGCGCAATAG
1101 TGTCACTGCTTCTAGCTCTGTGTCTGGCGGGGCTGATCAGCTGCCTTTCT1101 TGTCACTGCTTCTAGCTCTGTGTCTGGCGGGGCTGATCAGCTGCCTTTCT
1151 AA1151 AA
PcBLFYM2：PcBLFYM2:
0001 ATGGATCCTGAAGCATTCACGGCGAGCTTGTTTAAGTGGGACCCGCGTGG0001 ATGGATCCTGAAGCATTCACGGCGAGCTTGTTTAAGTGGGACCCGCGTGG
0051 GCTGGTGCCGCCACAGACCAGGGTGCTGGAACCGGTGGTGCCACTACCGG0051 GCTGGTGCCGCCACAGACCAGGGTGCTGGAACCGGTGGTGCCACTACCGG
0101 CAGCGATTTCCGCGGCCACCGCCTTCTCGGTGGTGCGTCCAAGGGAGCTA0101 CAGCGATTTCCGCGGCCACCGCCTTCTCGGTGGTGCGTCCAAGGGAGCTA
0151 GGTGGGCTGGAGGAGTTGTTCCAGGCTTATGGGATAAGGTACTACACGGC0151 GGTGGGCTGGAGGAGTTGTTCCAGGCTTATGGGATAAGGTACTACACGGC
0201 AGCGAAGATAGCGGAGCTTGGGTTCACGGTGAATACACTTGTGAATATGA0201 AGCGAAGATAGCGGAGCTTGGGTTCACGGTGAATACACTTGTGAATATGA
0251 AAGATGAGGAGCTTGATGAGATGATGAACAGCCTGTCTCAGATATTTAGG0251 AAGATGAGGAGCTTGATGAGATGATGAACAGCCTGTCTCAGATATTTAGG
0301 TGGGAACTCCTTGTTGGAGAGAGGTATGGTATTAAAGCTGCTGTTAGAGC0301 TGGGAACTCCTTGTTGGAGAGAGGTATGGTATTAAAGCTGCTGTTAGAGC
0351 TGAGAGGAGAAGGCTTGATGAGGAGGATTCACGAAGGCGTCACTTACTGT0351 TGAGAGGAGAAGGCTTGATGAGGAGGATTCACGAAGGCGTCACTTACTGT
0401 CTGGTGATACTACCAACTCTGTTGATGCTTTGTCCCAAGAAGGGTTATCT0401 CTGGTGATACTACCAACTCTGTTGATGCTTTGTCCCAAGAAGGGTTATCT
0451 GAGGAACCAGTGCAGCAAGAGAAGGAGGCAGCAGGGAGCGGAGGAGGAGG0451 GAGGAACCAGTGCAGCAAGAGAAGGAGGCAGCAGGGAGCGGAGGAGGAGG
0501 AACGTGGGACTTAGCGGTGGCAGTGACGGAGAGGAAGAAACAGCGGCGGA0501 AACGTGGGACTTAGCGGTGGCAGTGACGGAGAGGAAGAAACAGCGGCGGA
0551 GGAAGGGGCAAAGGAAGATAGTGGATGTTAATCACTATGATGAAAATGAA0551 GGAAGGGGCAAAGGAAGATAGTGGATGTTAATCACTATGATGAAAATGAA
0601 GACGATGAGAATGCTGAAGGCAGTGAGAGACAACGAGAGCACCCCTTCAT0601 GACGATGAGAATGCTGAAGGCAGTGAGAGACAACGAGAGCACCCCTTCAT
0651 AGTGACGGAGCCTGGTGAAGTGGCACGTGGCAAAAAAAACGGCCTTGATT0651 AGTGACGGAGCCTGGTGAAGTGGCACGTGGCAAAAAAAACGGCCTTGATT
0701 ACCTGTTCCATCTCTATGAGCAATGCCGTGATTTCTTGATCCAGGTCCAG0701 ACCTGTTCCATCTCTATGAGCAATGCCGTGATTTCTTGATCCAGGTCCAG
0751 AACATCGCCAAGGAGCGTGGCGAAAAATGCCCAACCAAGGTGACAAATCA0751 AACATCGCCAAGGAGCGTGGCGAAAAATGCCCAACCAAGGTGACAAATCA
0801 GGTGTTTAGGTATGCAAAGAAGGCTGGTGCAAGCTACATTAACAAGCCAA0801 GGTGTTTAGGTATGCAAAGAAGGCTGGTGCAAGCTACATTAACAAGCCAA
0851 AGATGAGACACTATGTGCACTGCTATGCGTTGCACTGCCTGGACGAGGAG0851 AGATGAGACACTATGTGCACTGCTATGCGTTGCACTGCCTGGACGAGGAG
0901 GCATCAGATGCACTGAGGAGGGTTTTCAAGGAAAGAGGAGAGAACGTGGG0901 GCATCAGATGCACTGAGGAGGGTTTTCAAGGAAAGAGGAGAGAACGTGGG
0951 GGCTTGGAGACAGGCTTGTTATAAGCCACTTGTGGGCATTGCAGCACGGC0951 GGCTTGGAGACAGGCTTGTTATAAGCCACTTGTGGGCATTGCAGCACGGC
1001 AAGGCTGGGATATTGATGCCATTTTCAATGCGCATCCTCGTCTGGCCATT1001 AAGGCTGGGATATTGATGCCATTTTCAATGCGCATCCTCGTCTGGCCATT
1051 TGGTATGTGCCCACGAAGCTGCGTCAACTTTGTCATGCTGAGCGCAATAG1051 TGGTATGTGCCCACGAAGCTGCGTCAACTTTGTCATGCTGAGCGCAATAG
1101 TGTCGCTGCTTCTAGCTCTGTGTCTGGCGGGGCTGATCAGCTGCCTTTCT1101 TGTCGCTGCTTCTAGCTCTGTGTCTGGCGGGGCTGATCAGCTGCCTTTCT
1151 AA1151 AA
PcBLFYF:PcBLFYF:
0001 ATGGATCCTGAAGCATTCACGGCGAGCTTGTTTAAGTGGGACCCGCGTGG0001 ATGGATCCTGAAGCATTCACGGCGAGCTTGTTTAAGTGGGACCCGCGTGG
0051 GCTGGTGCCGCCACAGACCAGGGTGCTGGAACCGGTGGTGCCACTACCGG0051 GCTGGTGCCGCCACAGACCAGGGTGCTGGAACCGGTGGTGCCACTACCGG
0101 CAGCGATTTCCGCGGCCACCGCCTTCTCGGTGGTGCGTCCAAGGGAGCTA0101 CAGCGATTTCCGCGGCCACCGCCTTCTCGGTGGTGCGTCCAAGGGAGCTA
0151 GGTGGGCTGGAGGAGTTGTTCCAGGCTTATGGGATAAGGTACTACACGGC0151 GGTGGGCTGGAGGAGTTGTTCCAGGCTTATGGGATAAGGTACTACACGGC
0201 AGCGAAGATAGCGGAGCTTGGGTTCACGGTGAATACACTTGTGAATATGA0201 AGCGAAGATAGCGGAGCTTGGGTTCACGGTGAATACACTTGTGAATATGA
0251 AAGATGAGGAGCTTGATGAGATGATGAACAGCCTGTCTCAGATATTTAGG0251 AAGATGAGGAGCTTGATGAGATGATGAACAGCCTGTCTCAGATATTTAGG
0301 TGGGAACTCCTTGTTGGAGAGAGGTATGGTATTAAAGCTGCTGTTAGAGC0301 TGGGAACTCCTTGTTGGAGAGAGGTATGGTATTAAAGCTGCTGTTAGAGC
0351 TGAGAGGAGAAGGCTTGATGAGGAGGATTCACGAAGGCGTCACTTACTGT0351 TGAGAGGAGAAGGCTTGATGAGGAGGATTCACGAAGGCGTCACTTACTGT
0401 CTGGTGATACTACCAACTCTGTTGATGCTTTGTCCCAAGAAGGGTTATCT0401 CTGGTGATACTACCAACTCTGTTGATGCTTTGTCCCAAGAAGGGTTATCT
0451 GAGGAACCAGTGCAGCAAGAGAAGGAGGCAGCAGGGAGCGGAGGAGGAGG0451 GAGGAACCAGTGCAGCAAGAGAAGGAGGCAGCAGGGAGCGGAGGAGGAGG
0501 AACGTGGGACTTAGCGGTGGCAGTGACGGAGAGGAAGAAACAGCGGCGGA0501 AACGTGGGACTTAGCGGTGGCAGTGACGGAGAGGAAGAAACAGCGGCGGA
0551 GGAAGGGGCAAAGGAAGATAGTGGATGTTAATCACTATGATGAAAATGAA0551 GGAAGGGGCAAAGGAAGATAGTGGATGTTAATCACTATGATGAAAATGAA
0601 GACGATGAGAATGCTGAAGGCAGTGAGAGACAACGAGAGCACCCCTTCAT0601 GACGATGAGAATGCTGAAGGCAGTGAGAGACAACGAGAGCACCCCTTCAT
0651 AGTGACGGAGCCTGGTGAAGTGGCACGTGGCAAAAAAAACGGCCTTGATT0651 AGTGACGGAGCCTGGTGAAGTGGCACGTGGCAAAAAAAACGGCCTTGATT
0701 ACCTGTTCCATCTCTATGAGCAATGCCGTGATTTCTTGATCCAGGTCCAG0701 ACCTGTTCCATCTCTATGAGCAATGCCGTGATTTCTTGATCCAGGTCCAG
0751 AACATCGCCAAGGAGCGTGGCGAAAAATGCCCAACCAAGGTGACAAATCA0751 AACATCGCCAAGGAGCGTGGCGAAAAATGCCCAACCAAGGTGACAAATCA
0801 GGTGTTTAGGTATGCAAAGAAGGCTGGTGCAAGCTACATTAACAAGCCAA0801 GGTGTTTAGGTATGCAAAGAAGGCTGGTGCAAGCTACATTAACAAGCCAA
0851 AGATGAGACACTATGTGCACTGCTATGCGTTGCACTGCCTGGACGAGGAG0851 AGATGAGACACTATGTGCACTGCTATGCGTTGCACTGCCTGGACGAGGAG
0901 GCATCAGATGCACTGAGGAGGGTTTTCAAGGAAAGAGGAGAGAACGTGGG0901 GCATCAGATGCACTGAGGAGGGTTTTCAAGGAAAGAGGAGAGAACGTGGG
0951 GGCTTGGAGACAGGCTTGTTATAAGCCACTTGTGGGCATTGCAGCACGGC0951 GGCTTGGAGACAGGCTTGTTATAAGCCACTTGTGGGCATTGCAGCACGGC
1001 AAGGCTGGGATATTGATGCCATTTTCAATGCGCATCCTCGTCTGGCCATT1001 AAGGCTGGGATATTGATGCCATTTTCAATGCGCATCCTCGTCTGGCCATT
1051 TGGTATGTGCCCACGAAGCTGCGTCAACTTTGTCATGCTGAGCGCAATAG1051 TGGTATGTGCCCACGAAGCTGCGTCAACTTTGTCATGCTGAGCGCAATAG
1101 TGTCACTGCTTCTAGCTCTGTGTCTGGCGGGGCTGATCAGCTGCCTTTCT1101 TGTCACTGCTTCTAGCTCTGTGTCTGGCGGGGCTGATCAGCTGCCTTTCT
1151 AA1151 AA
PcBLFYM3PcBLFYM3
0001 ATGGATCCTGAAGCATTCACGGCGAGCTTGTTTAAGTGGGACCCGCGTGG0001 ATGGATCCTGAAGCATTCACGGCGAGCTTGTTTAAGTGGGACCCGCGTGG
0051 GGTGGTGCCGCCACAGACCAGGGTGCTGGAACCGGTGGTGCCACTACCGG0051 GGTGGTGCCGCCACAGACCAGGGTGCTGGAACCGGTGGTGCCACTACCGG
0101 CAGCGATTTCCGCGGCCACCGCCTTCTCGGTGGTGCGTCCAAGGGAGCTA0101 CAGCGATTTCCGCGGCCACCGCCTTCTCGGTGGTGCGTCCAAGGGAGCTA
0151 GGTGGGCTGGAGGAGTTGTTCCAGGCTTATGGGATAAGGTACTACACGGC0151 GGTGGGCTGGAGGAGTTGTTCCAGGCTTATGGGATAAGGTACTACACGGC
0201 AGCGAAGATAGCGGAGCTTGGGTTCACGGTGAATACACTTGTGAATATGA0201 AGCGAAGATAGCGGAGCTTGGGTTCACGGTGAATACACTTGTGAATATGA
0251 AAGATGAGGAGCTTGATGAGATGATGAACAGCCTGTCTCAGATATTTAGG0251 AAGATGAGGAGCTTGATGAGATGATGAACAGCCTGTCTCAGATATTTAGG
0301 TGGGAACTCCTTGTTGGAGAGAGGTATGGTATTAAAGCTGCTGTTAGAGC0301 TGGGAACTCCTTGTTGGAGAGAGGTATGGTATTAAAGCTGCTGTTAGAGC
0351 TGAGAGGAGAAGGCTTGATGAGGAGGATTCACGAAGGCGTCACATACTGT0351 TGAGAGGAGAAGGCTTGATGAGGAGGATTCACGAAGGCGTCACATACTGT
0401 CTGGTGATACTACCAACTCTGTTGATGCTTTGTCCCAAGAAGGGTTATCT0401 CTGGTGATACTACCAACTCTGTTGATGCTTTGTCCCAAGAAGGGTTATCT
0451 GAGGAACCAGTGCAGCAAGAGAAGGAGGCAGCAGGGAGCGGAGGAGGAGG0451 GAGGAACCAGTGCAGCAAGAGAAGGAGGCAGCAGGGAGCGGAGGAGGAGG
0501 AACGTGGGACTTAGCGGTGGCAGTGACGGAGAGGAAGAAACAGCGGCGGA0501 AACGTGGGACTTAGCGGTGGCAGTGACGGAGAGGAAGAAACAGCGGCGGA
0551 GGAAGGGGCAAAGGAAGATAGTGGATGTTAATCACTATGATGAAAATGAA0551 GGAAGGGGCAAAGGAAGATAGTGGATGTTAATCACTATGATGAAAATGAA
0601 GATGATGAGAATGCTGAAGGCAGTGAGAGACAACGAGAGCACCCCTTCAT0601 GATGATGAGAATGCTGAAGGCAGTGAGAGACAACGAGAGCACCCCTTCAT
0651 AGTGACGGAGCCTGGTGAAGTGGCACGTGGCAAAAAAAACGGCCTTGATT0651 AGTGACGGAGCCTGGTGAAGTGGCACGTGGCAAAAAAAACGGCCTTGATT
0701 ACCTGTTCCATCTCTATGAGCAATGCCGTGATTTCTTGATCCAGGTCCAG0701 ACCTGTTCCATCTCTATGAGCAATGCCGTGATTTCTTGATCCAGGTCCAG
0751 AACATCGCCAAGGAGCGTGGCGAAAAATGCCCAACCAAGGTGACAAATCA0751 AACATCGCCAAGGAGCGTGGCGAAAAATGCCCAACCAAGGTGACAAATCA
0801 GGTGTTTAGGTATGCAAAGAAGGCTGGTGCAAGCTACATTAACAAGCCAA0801 GGTGTTTAGGTATGCAAAGAAGGCTGGTGCAAGCTACATTAACAAGCCAA
0851 AGATGAGACACTATGTGCACTGCTATGCGTTGCACTGCCTGGACGAGGAG0851 AGATGAGACACTATGTGCACTGCTATGCGTTGCACTGCCTGGACGAGGAG
0901 GCATCAGATGCACTGAGGAGGGTTTTCAAGGAAAGAGGAGAGAACGTGGG0901 GCATCAGATGCACTGAGGAGGGTTTTCAAGGAAAGAGGAGAGAACGTGGG
0951 GGCTTGGAGACAGGCTTGTTATAAGCCACTTGTGGGCATTGCAGCACGGC0951 GGCTTGGAGACAGGCTTGTTATAAGCCACTTGTGGGCATTGCAGCACGGC
1001 AAGGCTGGGATATTGATGCCATTTTCAATGCGCATCCTCGTCTGGCCATT1001 AAGGCTGGGATATTGATGCCATTTTCAATGCGCATCCTCGTCTGGCCATT
1051 TGGTATGTGCCCACGAAGCTGCGTCAACTTTGTCATGCTGAGCGCAATAG1051 TGGTATGTGCCCACGAAGCTGCGTCAACTTTGTCATGCTGAGCGCAATAG
1101 TGTCGCTGCTTCTAGCTCTGTGTCTGGCGGGGCTGATCAGCTGCCTTTCT1101 TGTCGCTGCTTCTAGCTCTGTGTCTGGCGGGGCTGATCAGCTGCCTTTCT
1151 AA1151 AA
根据以上的核苷酸序列，推测的氨基酸序列如下：According to the above nucleotide sequence, the deduced amino acid sequence is as follows:
PcBLFYM1：PcBLFYM1:
001 MDPEAFTASLFKWDPRGVVPPQTRVLEPVVPLPAAISAATAFSVVRPREL001 MDPEAFTASLFKWDPRGVVPPQTRVLEPVVPLPPAAISAATAFSVVRPREL
051 GGLEELFQAYGIRYYTAAKIAELGFTVNTLVNMKDEELDEMMNSLSQIFR051 GGLEELFQAYGIRYYTAAKIAELGFTVNTLVNMKDEELDEMMNSLSQIFR
101 WELLVGERYGIKAAVRAERRRLDEEDSRRRHILSGDTTNSVDALSQEGLS101 WELLVGERYGIKAAVRAERRRLDEEDSRRRRHILSGDTTNSVDALSQEGLS
151 EEPVQQEKEAAGSGGGGTWDLAVAVTERKKQRRRKGQRKIVDVNHYDENE151 EEPVQQEKEAAGSGGGGTWDLAVAVTERKKQRRRKGQRKIVDVNHYDENE
201 DDENAEGSERQREHPFIVTEPGEVARGKKNGLDYLFHLYEQCRDFLIQVQ201 DDENAEGSERQREHPFIVTEPGEVARGKKNGLDYLFHLYEQCRDFLIQVQ
251 NIAKERGEKCPTKVTNQVFRYAKKAGASYINKPKMRHYVHCYALHCLDEE251 NIAKERGEKCPTKVTNQVFRYAKKAGASYINKPKMRHYVHCYALHCLDEE
301 ASDALRRVFKERGENVGAWRQACYKPLVGIAARQGWDIDAIFNAHPRLAI301 ASDALRRVFKERGENVGAWRQACYKPLVGIAARQGWDIDAIFNAHPRLAI
351 WYVPTKLRQLCHAERNSVTASSSVSGGADQLPF351 WYVPTKLRQLCHAERNSVTASSSVSGGADQLPF
PcBLFYM2：PcBLFYM2:
001 MDPEAFTASLFKWDPRGLVPPQTRVLEPVVPLPAAISAATAFSVVRPREL001 MDPEAFTASLFKWDPRGLVPPQTRVLEPVVPLPPAAISAATAFSVVRPREL
051 GGLEELFQAYGIRYYTAAKIAELGFTVNTLVNMKDEELDEMMNSLSQIFR051 GGLEELFQAYGIRYYTAAKIAELGFTVNTLVNMKDEELDEMMNSLSQIFR
101 WELLVGERYGIKAAVRAERRRLDEEDSRRRHLLSGDTTNSVDALSQEGLS101 WELLVGERYGIKAAVRAERRRLDEEDSRRRRHLLSGDTTNSVDALSQEGLS
151 EEPVQQEKEAAGSGGGGTWDLAVAVTERKKQRRRKGQRKIVDVNHYDENE151 EEPVQQEKEAAGSGGGGTWDLAVAVTERKKQRRRKGQRKIVDVNHYDENE
201 DDENAEGSERQREHPFIVTEPGEVARGKKNGLDYLFHLYEQCRDFLIQVQ201 DDENAEGSERQREHPFIVTEPGEVARGKKNGLDYLFHLYEQCRDFLIQVQ
251 NIAKERGEKCPTKVTNQVFRYAKKAGASYINKPKMRHYVHCYALHCLDEE251 NIAKERGEKCPTKVTNQVFRYAKKAGASYINKPKMRHYVHCYALHCLDEE
301 ASDALRRVFKERGENVGAWRQACYKPLVGIAARQGWDIDAIFNAHPRLAI301 ASDALRRVFKERGENVGAWRQACYKPLVGIAARQGWDIDAIFNAHPRLAI
351 WYVPTKLRQLCHAERNSVAASSSVSGGADQLPF351 WYVPTKLRQLCHAERNSVAASSSVSGGADQLPF
PcBLFYF:PcBLFYF:
001 MDPEAFTASLFKWDPRGLVPPQTRVLEPVVPLPAAISAATAFSVVRPREL001 MDPEAFTASLFKWDPRGLVPPQTRVLEPVVPLPPAAISAATAFSVVRPREL
051 GGLEELFQAYGIRYYTAAKIAELGFTVNTLVNMKDEELDEMMNSLSQIFR051 GGLEELFQAYGIRYYTAAKIAELGFTVNTLVNMKDEELDEMMNSLSQIFR
101 WELLVGERYGIKAAVRAERRRLDEEDSRRRHLLSGDTTNSVDALSQEGLS101 WELLVGERYGIKAAVRAERRRLDEEDSRRRRHLLSGDTTNSVDALSQEGLS
151 EEPVQQEKEAAGSGGGGTWDLAVAVTERKKQRRRKGQRKIVDVNHYDENE151 EEPVQQEKEAAGSGGGGTWDLAVAVTERKKQRRRKGQRKIVDVNHYDENE
201 DDENAEGSERQREHPFIVTEPGEVARGKKNGLDYLFHLYEQCRDFLIQVQ201 DDENAEGSERQREHPFIVTEPGEVARGKKNGLDYLFHLYEQCRDFLIQVQ
251 NIAKERGEKCPTKVTNQVFRYAKKAGASYINKPKMRHYVHCYALHCLDEE251 NIAKERGEKCPTKVTNQVFRYAKKAGASYINKPKMRHYVHCYALHCLDEE
301 ASDALRRVFKERGENVGAWRQACYKPLVGIAARQGWDIDAIFNAHPRLAI301 ASDALRRVFKERGENVGAWRQACYKPLVGIAARQGWDIDAIFNAHPRLAI
351 WYVPTKLRQLCHAERNSVTASSSVSGGADQLPF351 WYVPTKLRQLCHAERNSVTASSSVSGGADQLPF
PcBLFYM3PcBLFYM3
001 MDPEAFTASLFKWDPRGVVPPQTRVLEPVVPLPAAISAATAFSVVRPREL001 MDPEAFTASLFKWDPRGVVPPQTRVLEPVVPLPPAAISAATAFSVVRPREL
051 GGLEELFQAYGIRYYTAAKIAELGFTVNTLVNMKDEELDEMMNSLSQIFR051 GGLEELFQAYGIRYYTAAKIAELGFTVNTLVNMKDEELDEMMNSLSQIFR
101 WELLVGERYGIKAAVRAERRRLDEEDSRRRHILSGDTTNSVDALSQEGLS101 WELLVGERYGIKAAVRAERRRLDEEDSRRRRHILSGDTTNSVDALSQEGLS
151 EEPVQQEKEAAGSGGGGTWDLAVAVTERKKQRRRKGQRKIVDVNHYDENE151 EEPVQQEKEAAGSGGGGTWDLAVAVTERKKQRRRKGQRKIVDVNHYDENE
201 DDENAEGSERQREHPFIVTEPGEVARGKKNGLDYLFHLYEQCRDFLIQVQ201 DDENAEGSERQREHPFIVTEPGEVARGKKNGLDYLFHLYEQCRDFLIQVQ
251 NIAKERGEKCPTKVTNQVFRYAKKAGASYINKPKMRHYVHCYALHCLDEE251 NIAKERGEKCPTKVTNQVFRYAKKAGASYINKPKMRHYVHCYALHCLDEE
301 ASDALRRVFKERGENVGAWRQACYKPLVGIAARQGWDIDAIFNAHPRLAI301 ASDALRRVFKERGENVGAWRQACYKPLVGIAARQGWDIDAIFNAHPRLAI
351 WYVPTKLRQLCHAERNSVAASSSVSGGADQLPF351 WYVPTKLRQLCHAERNSVAASSSVSGGADQLPF
本发明利用雌株、雄株和雌雄同株植物在RNA 或DNA序列上的单碱基序列差异，进行黄连木植株性别鉴定。The present invention utilizes the single base sequence differences in RNA or DNA sequences of female plants, male plants and monoecious plants to identify the sex of Pistacia chinensis plants.
本发明用于创建早开花植物基因工程株系。The invention is used for creating genetically engineered strains of early flowering plants.
本发明的特征在于利用黄连木开花主控基因PcBLFY基因构建植物表达载体，通过转基因获得早开花的植株。The present invention is characterized in that the flowering main control gene PcBLFY of Pistacia chinensis is used to construct the plant expression vector, and the early flowering plants are obtained through transgene.
本发明克隆了黄连木雌株、雄株和雌雄同株植株的LEAFY基因，不同性别的植株间一些位点存在单碱基序列多态性，可以以此为基础，建立黄连木苗期的性别鉴定技术。The present invention clones the LEAFY gene of the female plant, the male plant and the monoecious plant of Pistacia chinensis, and there are single base sequence polymorphisms in some sites among the plants of different sexes, which can be used as a basis to establish the sex of Pistacia chinensis seedling stage identification technology.
附图说明： Description of drawings:
图1为RT-PCR结果；Fig. 1 is RT-PCR result;
图2为LEAFY基因5’RACE电泳图；Figure 2 is the 5' RACE electrophoresis of the LEAFY gene;
图3为LEAFY基因3’RACE电泳图；Figure 3 is a 3' RACE electrophoresis diagram of the LEAFY gene;
图4为LEAFY 基因的编码区PCR扩增产物电泳图；Fig. 4 is the electrophoresis diagram of the PCR amplification product of the coding region of the LEAFY gene;
图5为PcBLFYF基因蛋白的SDS-PAGE电泳图；Fig. 5 is the SDS-PAGE electrophoresis figure of PcBLFYF gene protein;
图6为pCPcBLFY载体构建，图中：1.目的基因；2. Marker(DL2000plus)；3和4. pCPcBLFY/pC1305.1重组质粒；5. Marker(Trans 15K)；6. 重组质粒酶切产物BglII/BstEII；Figure 6 is the pCPcBLFY vector construction, in the figure: 1. Target gene; 2. Marker (DL2000plus); 3 and 4. pCPcBLFY/pC1305.1 recombinant plasmid; 5. Marker (Trans 15K); 6. Recombinant plasmid digestion product BglII /BstEII;
图7为pCPcBLFY植物表达载体图。Fig. 7 is a map of the plant expression vector of pCPcBLFY.
具体实施方式 Detailed ways
下面结合实施例及附图对本发明作进一步的描述，但本发明的实施方式不限于此。接续说明书中所描述的黄连木开花基因（PcBLFY）及其应用的全部内容，进行下列实施例1-4。The present invention will be further described below in conjunction with the embodiments and drawings, but the embodiments of the present invention are not limited thereto. Continuing the entire content of Pistacia flowering gene (PcBLFY) and its application described in the specification, the following Examples 1-4 were carried out.
实施例1：Example 1:
根据已克隆的模式植物LEAFY基因保守序列设计兼并引物（正向引物：5′-GARGTGGCACGTGGSAARAARAA-3′；反向引物：5′-CGGAGYTTGGTGGGMACRTACCAAccording to the conserved sequence of cloned model plant LEAFY gene, degenerate primers were designed (forward primer: 5′-GARGTGGCACGTGGSAARAARAA-3′; reverse primer: 5′-CGGAGYTTGGTGGGMACRTACCA
-3′），以黄连木雌株、雄株、雌雄同株植物总RNA为模板，进行RT-PCR扩增，获得了一段361bp的cDNA序列（图1），并分别以2个基因特异性引物GSP1-1(引物序列为5’-CCTTGAAAACCCTCCTCAGTGCATCTGA-3’)和NGSP1-2(引物序列为5’- CGCCACGCTCCTTGGCGATGTTCTG-3’)进行5’RACE巢式扩增，获得960bp的cDNA片段（图2）；同时利用GSP2-3(引物序列为5’-CAGGTCCAGAACATCGCCAAGGAGC-3’)和NGSP2-1(引物序列为5’-ATGCCCAACCAAGGTGACAAATCAGG-3’)两个基因特异性引物进行3’RACE巢式扩增获得620bp的扩增片段（图3），拼接后得到的cDNA全长序列为1570bp.-3'), using the total RNA of Pistacia chinensis female, male, and monoecious plants as templates, RT-PCR amplification was performed, and a 361bp cDNA sequence was obtained (Figure 1), and two gene-specific Primers GSP1-1 (primer sequence is 5'-CCTTGAAAACCCTCTCAGTGCATCTGA-3') and NGSP1-2 (primer sequence is 5'-CGCCACGCTCCTTGGCGATGTTCTG-3') were used for 5' RACE nested amplification to obtain a 960bp cDNA fragment (Figure 2) ; At the same time, two gene-specific primers of GSP2-3 (the primer sequence is 5'-CAGGTCCAGAACATCGCCAAGGAGC-3') and NGSP2-1 (the primer sequence is 5'-ATGCCCAACCAAGGTGACAAATCAGG-3') were used for 3'RACE nested amplification to obtain 620bp The amplified fragment (Figure 3), the full-length cDNA sequence obtained after splicing is 1570bp.
根据获得的cDNA，设计编码区扩增PCR引物（正向引物序列：5’-GCTCAAAATCTCAGTTGCTCAG-3’；反向引物序列：5’-TCGGTCATTCAGTGAGGCT TT-3’），分别以不同性别黄连木植株的总RNA为模板，进行RT-PCR，在雄株、雌株、雌雄同株植物中分别获得2种（PcBLFYM1，PcBLFYM2）、1种（PcBLFYF）、2种（PcBLFYF,PcBLFYM3）类型的LEAFY基因（图4），这4种类型的基因在四个位点上有不同程度的差异，可以根据这些差异设计用于鉴定三种类型植株的PCR反应。According to the obtained cDNA, PCR primers for amplification of the coding region were designed (forward primer sequence: 5'-GCTCAAAATCTCAGTTGCTCAG-3'; reverse primer sequence: 5'-TCGGTCATTCAGTGAGGCT TT-3'), and the total RNA was used as a template, and RT-PCR was performed to obtain 2 types of LEAFY genes (PcBLFYM1, PcBLFYM2), 1 type (PcBLFYF), and 2 types (PcBLFYF, PcBLFYM3) in male plants, female plants, and monoecious plants, respectively (Fig. 4), the four types of genes have different degrees of differences in the four sites, and PCR reactions for identifying the three types of plants can be designed based on these differences.
实施例2：Example 2:
利用从编码区ATG开始的第394位、603位碱基序列的差异（但不限于此两个位点），设计以394位碱基为3’端末端碱基的正向引物和以603位碱基为反向引物3’端末端碱基的PCR扩增引物，用于鉴定植株的性别：Using the difference between the 394th and 603rd base sequences starting from ATG in the coding region (but not limited to these two positions), design the forward primer with the 394th base as the 3' terminal base and the 603rd base The base is the PCR amplification primer of the 3' end base of the reverse primer, which is used to identify the sex of the plant:
引物394F1：5’-GAGGATTCACGAAGGCGTCACA-3’Primer 394F1: 5'-GAGGATTCACGAAGGCGTCACA-3'
引物394F2：5’-GAGGATTCACGAAGGCGTCACT-3’Primer 394F2: 5'-GAGGATTCACGAAGGCGTCACT-3'
引物 603R1：5’-CTGCCTTCAGCATTCTCATCG-3’Primer 603R1: 5'-CTGCCTTCAGCATTCTCATCG-3'
引物 603R2：5’-CTGCCTTCAGCATTCTCATCA-3’Primer 603R2: 5'-CTGCCTTCAGCATTCTCATCA-3'
利用以上两对引物，可进行黄连木植株性别鉴定，如下表:Utilize above two pairs of primers, can carry out sex identification of Pistacia chinensis plant, as following table:
表中+表示可获得PCR扩增产物（如通过RT-PCR可获得251bp的cDNA扩增片段），-表示PCR反应为阴性。In the table, + indicates that the PCR amplification product can be obtained (for example, a 251bp cDNA amplification fragment can be obtained by RT-PCR), - indicates that the PCR reaction is negative.
实施例3：Example 3:
设计引物（正向引物序列：5’-GGAATTCCATATGGATCCTGAAGCATTCA-3’；反向引物序列：5’-CCCAAGCTTGGTCATTCAGTGAGGCTTTAAT-3’）扩增PcBLFYF的编码区序列，利用双酶切 NdeI/HindⅢ 插入pET28a原核表达载体，构建的重组载体导入BL21菌株中进行表达，获得大小为43kD的蛋白（图5）。Primers were designed (forward primer sequence: 5'-GGAATTCCATATGGATCCTGAAGCATTCA-3'; reverse primer sequence: 5'-CCCAAGCTTGGTCATTCAGTGAGGCTTTAAT-3') to amplify the coding region sequence of PcBLFYF, and inserted into the pET28a prokaryotic expression vector by double enzyme cutting NdeI/HindⅢ. The constructed recombinant vector was introduced into BL21 strain for expression, and a protein with a size of 43kD was obtained (Figure 5).
实施例4：Example 4:
设计引物（正向引物序列：5’-GAAGATCTGATGGATCCTGAAGCATTC-3’；反向引物序列：5’-GGGGTCACCTTAGAAAGGCAGCTGATC-3’）扩增PcBLFYF的编码区序列，利用双酶切BglⅡ/BstEⅡ将其插入到植物表达载体pCAMBIA1305.1中，构建的植物表达重组载体pCPcBLFY用于转化童期长的植物，使其提早开花结果（图6和7）。Design primers (forward primer sequence: 5'-GAAGATCTGATGGATCCTGAAGCATTC-3'; reverse primer sequence: 5'-GGGGTCACCTTAGAAAGGCAGCTGATC-3') to amplify the coding region sequence of PcBLFYF, and insert it into plant expression by double enzyme cutting BglⅡ/BstEⅡ In the vector pCAMBIA1305.1, the constructed plant expression recombinant vector pCPcBLFY was used to transform plants with long juvenile stages to make them flower and bear fruit earlier (Figures 6 and 7).
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103288942A (en) * | 2013-05-02 | 2013-09-11 | 东北林业大学 | African agapanthus flowering gene ApFT protein and coding gene and probe thereof |
| CN103667313A (en) * | 2013-12-05 | 2014-03-26 | 浙江农林大学 | Method for cloning complete sequence of coding region of homologous gene of LFY in plum blossom |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103288942A (en) * | 2013-05-02 | 2013-09-11 | 东北林业大学 | African agapanthus flowering gene ApFT protein and coding gene and probe thereof |
| CN103667313A (en) * | 2013-12-05 | 2014-03-26 | 浙江农林大学 | Method for cloning complete sequence of coding region of homologous gene of LFY in plum blossom |
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