技术领域technical field
本发明涉及一种快速鉴别杂交水牛染色体核型的方法。The invention relates to a method for quickly identifying the karyotype of hybrid buffalo chromosomes.
背景技术Background technique
亚洲水牛包括河流型(RiverBuffalo)和沼泽型(SwampBuffalo)两个亚种。河流型水牛主要产于南亚地区，例如尼里-拉菲水牛(巴基斯坦)和摩拉水牛(印度)，沼泽型水牛则以我国为最。水牛具有较强的抗病力和耐粗饲的特点，适应湿热气候，非常适合我国南方地区饲养，南方地区鲜奶消费的缺口也可以由水牛奶填补。水牛奶因口感独特，奶香味浓郁，其脂肪、蛋白质和钙、磷的含量比普通牛奶高而备受到人们的青睐，从而激起了奶水牛业的兴起和快速发展。这些年来，在国家产业政策刺激下，水牛奶业日益受到重视，在不久的将来，奶水牛行业必将成为南方奶业的重要支柱。但是水牛属于单胎动物，繁殖力低，与黄牛相比，水牛具有发情期晚、发情不明显、受胎率低及产后发情间隔时间长等特点，整体繁殖力低(王根林，2006)。水牛产奶量与荷斯坦牛相比，仅为其一半，沼泽型水牛的产奶量更低。在现代化养殖的条件下，仅靠自然繁殖是远远不能满足需要的，因此一般都会引进河流型水牛进行杂交以提高水牛的繁殖性能和产奶量。河流型水牛染色体数目是50(DuttMK，BhattacharyaP.ChromosomesoftheIndianwaterBuffalo.Nature，1952，27:1129-1129)，而沼泽型水牛为48(BerardinoDD，IannuzziL.ChromosomebandinghomologiesinSwampandMurrahBuffalo.TheJournalofHeredity，1981，72:183-188)。导致两者染色体数目不同的原因是，沼泽型水牛最大的1号染色体，基因和形态上与河流型水牛4和9号染色体极为相似，相当于河流型水牛第4号染色体短臂的端粒和9号染色体的着丝粒串联融合，形成一条染色体(BerardinoDD，IannuzziL.ChromosomebandinghomologiesinSwampandMurrahBuffalo.TheJournalofHeredity，1981，72:183-188)。两个亚种之间会杂交产生染色体为49的后代，这些后代之间是可育的，相互杂交会产生48、49和50三种不同染色体数的后代。根据有关的研究表明，杂交水牛的繁殖性能比纯种有所降低，主要体现在其核型数2n＝49的杂交水牛会产生异常配子，导致胚胎发育的不正常，最终导致胚胎在早期便死亡，宏观上表现为情期配种受胎率和年受胎率分别降低12.3％和6.4％，产仔间隔长达97.6天(黄右军，尚江华，梁梦玫，张秀芳，黄芬香.河流型水牛与沼泽型水牛杂交后代(2n＝49)染色体遗传与繁殖力的研究.遗传，2003，25(2)：155-159)。若通过核型水平对水牛进行选择，那么对杂交水牛群的整体繁殖能力将有显著的提升，辨别杂交水牛核型数也显得非常重要。Asian buffalo includes two subspecies of river type (RiverBuffalo) and swamp type (SwampBuffalo). River buffaloes are mainly produced in South Asia, such as Niri-Rafi buffaloes (Pakistan) and Mora buffaloes (India), while swamp buffaloes are most common in my country. Buffaloes have strong disease resistance and resistance to rough feeding. They adapt to hot and humid climates and are very suitable for breeding in southern China. The gap in fresh milk consumption in southern China can also be filled by buffalo milk. Buffalo milk is favored by people because of its unique taste, strong milk flavor, and higher content of fat, protein, calcium and phosphorus than ordinary milk, which has stimulated the rise and rapid development of the milk buffalo industry. In recent years, under the stimulation of the national industrial policy, the buffalo milk industry has been paid more and more attention. In the near future, the milk buffalo industry will become an important pillar of the southern dairy industry. However, buffaloes are single-born animals with low fecundity. Compared with cattle, buffaloes have the characteristics of late estrus, inconspicuous estrus, low conception rate, and long postpartum estrus interval, and their overall fecundity is low (Wang Genlin, 2006). The milk yield of buffalo is only half of that of Holstein cattle, and the milk yield of swamp buffalo is even lower. Under the conditions of modern breeding, natural reproduction alone is far from enough to meet the needs. Therefore, river buffaloes are generally introduced for crossbreeding to improve the reproductive performance and milk production of buffaloes. The number of chromosomes in river buffalo is 50 (DuttMK, BhattacharyaP.ChromosomesoftheIndianwaterBuffalo.Nature, 1952, 27:1129-1129), while that in swamp buffaloes is 48 (BerardinoDD, IannuzziL.ChromosomebandinghomologiesinSwampandMurrahBuffalo.TheJournalofHeredity, 83:1981, 7 The reason for the difference in the number of chromosomes between the two is that the largest chromosome 1 of the swamp buffalo is very similar in gene and morphology to chromosomes 4 and 9 of the river buffalo, which is equivalent to the telomere and the short arm of chromosome 4 of the river buffalo. The centromere of chromosome 9 fused in tandem to form a single chromosome (Berardino DD, Iannuzzi L. Chromosome banding homologies in Swamp and Murrah Buffalo. The Journal of Heredity, 1981, 72: 183-188). Crossbreeding between two subspecies will produce offspring with chromosome 49, which are fertile, and crossbreeding with each other will produce offspring with three different chromosome numbers of 48, 49 and 50. According to related studies, the reproductive performance of hybrid buffaloes is lower than that of purebred buffaloes, which is mainly reflected in the fact that hybrid buffaloes with karyotype 2n=49 will produce abnormal gametes, which will lead to abnormal embryo development and eventually cause the embryos to die at an early stage , macroscopically, the conception rate of mating in estrus and annual conception rate decreased by 12.3% and 6.4% respectively, and the calving interval was as long as 97.6 days (Huang Youjun, Shang Jianghua, Liang Mengmei, Zhang Xiufang, Huang Fenxiang. The offspring of crossbreeding of river buffalo and swamp type buffalo (2n=49) Studies on Chromosomal Inheritance and Fecundity. Heredity, 2003, 25(2): 155-159). If the buffaloes are selected at the karyotype level, the overall reproductive capacity of the hybrid buffalo population will be significantly improved, and it is also very important to identify the karyotype numbers of the hybrid buffaloes.
传统鉴定核型的方法是采用取外周血淋巴细胞培养一段时间后，加入秋水仙素使细胞分裂停留在中期，再低渗固定，制作滴片，通过染色体照片的对比分析对染色体数目进行分组，观测和描述组内各染色体形态和特征，进而确定其染色体组型并阐明生物的染色体组成。但是该方法较为繁琐，周期长，费时费力，成本也不低，检测大群体杂交水牛便非常麻烦，不符合现代化大规模养殖的发展方向。然而快速检测杂交水牛核型的方法依旧没有相关文献证明已经正在研究或者研究出来，甚至连这方面的探索也没有文献资料可供查询。哺乳动物中，亚种间由于长时间的地理和生殖隔离，相同的基因也会有不同的突变和演化。在水牛的亚种杂交中，不同核型的杂交水牛在特定染色体上的基因会有所区别，这些区别会体现在碱基序列上。因此本研究依据分子标记的应用和杂交水牛核型分析，将二者联系起来，建立利用分子标记快速检测杂交水牛核型的方法，该方法尚无相关文献发表或说明，属于创新型研究，可快速方便大规模检测杂交水牛的核型，改良群体结构，从而提高繁殖和泌乳性能，加快杂交水牛的品种改良速度。The traditional method of identifying karyotype is to take peripheral blood lymphocytes and culture them for a period of time, then add colchicine to stop the cell division in the middle stage, then fix them in low osmosis, make drops, and group the number of chromosomes by comparing and analyzing chromosome photos. Observe and describe the morphology and characteristics of each chromosome in the group, and then determine its karyotype and clarify the chromosome composition of organisms. However, this method is cumbersome, long-term, time-consuming and labor-intensive, and the cost is not low. It is very troublesome to detect large groups of hybrid buffaloes, which does not conform to the development direction of modern large-scale breeding. However, there is still no relevant literature to prove that the method of quickly detecting the karyotype of hybrid buffalo has been studied or developed, and even the exploration in this area has no literature for query. In mammals, due to long-term geographical and reproductive isolation between subspecies, the same gene will have different mutations and evolutions. In the hybridization of buffalo subspecies, the genes of hybrid buffaloes with different karyotypes on specific chromosomes will be different, and these differences will be reflected in the base sequence. Therefore, based on the application of molecular markers and hybrid buffalo karyotype analysis, this study combined the two to establish a method for rapid detection of hybrid buffalo karyotypes using molecular markers. This method has not been published or explained in relevant literature, and it is an innovative research. Quickly and conveniently detect the karyotype of hybrid buffaloes on a large scale, improve the population structure, thereby improving reproductive and lactating performance, and speeding up the breed improvement of hybrid buffaloes.
分子标记技术应用非常广泛，尤其在亚种的鉴别上，亚种间由于长时间的地理和生殖隔离，相同的基因也会有不同的突变和演化通过分子标记便可找出这些差异，加以辨别。敖光辉等人利用SSR对籼粳稻亚种之间进行标记差异，发现差异明显(敖光辉，王丽，魏琴，周黎军.釉粳稻亚种间分子标记差异分析.安徽农业科学，2008，36(5):1778-1780)；张晓璐利用分子标记在虎亚种识别中达到极高的准确率(张晓璐.虎亚种识别的分子遗传学技术的研究.[硕士学位].哈尔滨：东北林业大学图书馆，2005)，可见分子标记在鉴别亚种方面是非常方便准确，具有极大的优势。不同核型的杂交水牛在特定染色体(河流型4和9号染色体，沼泽型1号染色体)的数量上有所区别，2n＝48核型的水牛拥有两条沼泽型1号染色体，无河流型4和9号染色体；2n＝49核型的水牛拥有一条沼泽型1号染色体，河流型4和9号染色体个一条；2n＝50核型的水牛无沼泽型1号染色体，拥有两条河流型4和9号染色体。这些区别会导致位于这些染色体上的基因序列，在不同核型杂交水牛上会有差异，利用分子标记方法，可以找到不同核型之间基因序列的稳定性差异，从而辨别核型。经过大量的文献查找，有如下基因位于以上3条染色体中：ACO2、ACTA1、CSF1R、CSF2、CGN1、GAPDH、GLI、IFNG、IGF1、RBP3、LDHB、LYZ、TPI1、LDLR、EEF2。其中，后两个在河流型水牛9号染色体上(IannuzziL，Cnriabbam，ViaA.AgeneticphysicalmapinriverBuffalo(Bubalusbubalis，2n＝50).Caryologia，1998，5:311-318；NahasSE，HondtHA，SoussaSF，GhorAE，HanssanAA.AssignmentofnewlocitoriverBuffalochromosomesconfirmstheNatureofchromosomes4and5.J.Anim.Breed.Genet，1999，116:21-28)。通过大量的试验最终从RBP3基因上找到符合条件的突变位点，可以较为准确快速地辨别不同核型的杂交水牛。Molecular marker technology is widely used, especially in the identification of subspecies. Due to long-term geographical and reproductive isolation between subspecies, the same gene will also have different mutations and evolution. These differences can be found and identified through molecular markers. . Ao Guanghui et al. used SSR to detect marker differences between indica and japonica rice subspecies, and found significant differences (Ao Guanghui, Wang Li, Wei Qin, Zhou Lijun. Analysis of molecular marker differences among glaze japonica rice subspecies. Anhui Agricultural Sciences, 2008, 36( 5): 1778-1780); Zhang Xiaolu used molecular markers to achieve extremely high accuracy in tiger subspecies identification (Zhang Xiaolu. Research on molecular genetics techniques for tiger subspecies identification. [Master's degree]. Harbin: Northeast Forestry University Books Guan, 2005), it can be seen that molecular markers are very convenient and accurate in identifying subspecies, and have great advantages. Hybrid buffaloes with different karyotypes differ in the number of specific chromosomes (river type chromosomes 4 and 9, swamp type chromosome 1), buffaloes with 2n=48 karyotype have two swamp type chromosomes 1, and no river type Chromosomes 4 and 9; buffaloes with 2n=49 karyotype have one swamp-type chromosome 1, and river-type chromosomes 4 and 9; buffaloes with 2n=50 karyotype have no swamp-type chromosome 1 and have two river-type chromosomes Chromosomes 4 and 9. These differences will lead to differences in gene sequences located on these chromosomes in hybrid buffaloes with different karyotypes. Using molecular marker methods, the stability differences of gene sequences between different karyotypes can be found to distinguish karyotypes. After a lot of literature search, the following genes are located in the above three chromosomes: ACO2, ACTA1, CSF1R, CSF2, CGN1, GAPDH, GLI, IFNG, IGF1, RBP3, LDHB, LYZ, TPI1, LDLR, EEF2.其中，后两个在河流型水牛9号染色体上(IannuzziL，Cnriabbam，ViaA.AgeneticphysicalmapinriverBuffalo(Bubalusbubalis，2n＝50).Caryologia，1998，5:311-318；NahasSE，HondtHA，SoussaSF，GhorAE，HanssanAA.AssignmentofnewlocitoriverBuffalochromosomesconfirmstheNatureofchromosomes4and5 . J. Anim. Breed. Genet, 1999, 116:21-28). Through a large number of experiments, the qualified mutation sites were finally found from the RBP3 gene, which can accurately and quickly distinguish the hybrid buffaloes with different karyotypes.
发明内容Contents of the invention
本发明的目的在于建立一种快速鉴别杂交水牛染色体核型的新方法。The purpose of the invention is to establish a new method for quickly identifying the karyotype of hybrid buffalo chromosomes.
通过优化外周血淋巴细胞培养方法获得杂交水牛的染色体数目，选取定位于沼泽型水牛1号染色体上的RBP3基因进行扩增测序，找到特异性突变位点，与染色体数目进行对比，通过凝胶的条带的区别来鉴别染色体数量的多少。The number of chromosomes of hybrid buffalo was obtained by optimizing the culture method of peripheral blood lymphocytes, and the RBP3 gene located on chromosome 1 of swamp type buffalo was selected for amplification and sequencing to find the specific mutation site and compared with the number of chromosomes. The difference in bands is used to identify the number of chromosomes.
以黄牛的DNA为模板设计引物，得到可辨别核型的水牛RBP3基因的两个突变位点，它的核苷酸序列如序列表SEQIDNO：1和SEQIDNO：2所示。RBP3-1的365bp处有一个A365-T365的碱基突变；在RBP3-2的442bp处有一个G442-A442的碱基突变，两者可以辨别杂交水牛的三种核型。Primers are designed using the DNA of cattle as a template to obtain two mutation sites of the buffalo RBP3 gene that can distinguish the karyotype, and its nucleotide sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence table. There is a base mutation of A365-T365 at 365bp of RBP3-1; there is a base mutation of G442-A442 at 442bp of RBP3-2, and the two can distinguish the three karyotypes of hybrid buffaloes.
设计了可以辨别核型的RBP3基因的两段DNA片段的引物对，所述的RBP3-1引物对的正向引物为5’-GTGCCGCTGCTACTCTCTTACT-3’(SEQIDNO：3)，反向引物为5’-TCCTTACCTATGTGACGCTTGACG-3(SEQIDNO：4)；所述的RBP3-2引物对的正向引物为5’-AACACCTATCCACGACCTTTATCAT-3’(SEQIDNO：5)，反向引物为5’-AGTTGCTTTAGCGAAGACACTATTATC-3’(SEQIDNO：6)。A primer pair of two DNA fragments of the RBP3 gene that can distinguish the karyotype is designed, the forward primer of the RBP3-1 primer pair is 5'-GTGCCGCTGCTACTCTCTTACT-3' (SEQ ID NO: 3), and the reverse primer is 5' -TCCTTACCTATGTGACGCTTGACG-3 (SEQ ID NO: 4); the forward primer of the RBP3-2 primer pair is 5'-AACACCTATCCACGACCTTTATCAT-3' (SEQ ID NO: 5), and the reverse primer is 5'-AGTTGCTTTAGCGAAGACACTATTATC-3' (SEQ ID NO: 6).
一种快速鉴别杂交水牛染色体核型的方法，包括以下步骤：A method for quickly identifying the karyotype of a hybrid buffalo, comprising the following steps:
1)提取杂交水牛的总DNA；1) extracting the total DNA of hybrid buffalo;
2)根据水牛RBP3基因的两个突变位点的核苷酸序列，设计两组引物对——RBP3-1引物对和RBP3-2引物对，其中：2) According to the nucleotide sequences of the two mutation sites of the buffalo RBP3 gene, two sets of primer pairs are designed—the RBP3-1 primer pair and the RBP3-2 primer pair, wherein:
RBP3-1的正向引物为5’-GTGCCGCTGCTACTCTCTTACT-3’，The forward primer of RBP3-1 is 5'-GTGCCGCTGCTACTCTCTTACT-3',
RBP3-1的反向引物为5’-TCCTTACCTATGTGACGCTTGACG-3’；The reverse primer of RBP3-1 is 5'-TCCTTACCTATGTGACGCTTGACG-3';
RBP3-2的正向引物为5’-AACACCTATCCACGACCTTTATCAT-3’，The forward primer of RBP3-2 is 5'-AACACCTATCCACGACCTTTATCAT-3',
RBP3-2的反向引物为5’-AGTTGCTTTAGCGAAGACACTATTATC-3’；The reverse primer of RBP3-2 is 5'-AGTTGCTTTAGCGAAGACACTATTATC-3';
3)分别用步骤2)中的两组引物对杂交水牛的DNA进行PCR扩增，得到两种扩增产物；3) carry out PCR amplification to the DNA of hybrid buffalo with two groups of primers in step 2) respectively, obtain two kinds of amplification products;
4)分别对步骤3)所得到的两种扩增产物进行酶切、分型：RBP3-1引物对的扩增产物选用Ecil内切酶，酶切条带中出现2条带确认为2N＝50核型；RBP3-2引物对的扩增产物选用Drdl内切酶，酶切条带中出现1条带确认为2N＝48核型，酶切条带中出现3条带确认为2N＝49核型。4) Digest and type the two amplified products obtained in step 3) respectively: the amplified product of the RBP3-1 primer pair is selected with Ecil endonuclease, and 2 bands appear in the digested bands, which are confirmed as 2N= 50 karyotype; the amplified product of RBP3-2 primer pair was selected with Drdl endonuclease, 1 band appeared in the enzyme-digested band was confirmed as 2N=48 karyotype, and 3 bands appeared in the enzyme-digested band were confirmed as 2N=49 Karyotype.
步骤3)中的PCR扩增体系为：双蒸水8μL、Mix10μL、正向引物0.5μL、反向引物0.5μL、杂交水牛DNA1μL。The PCR amplification system in step 3) is: 8 μL of double distilled water, 10 μL of Mix, 0.5 μL of forward primer, 0.5 μL of reverse primer, and 1 μL of hybrid buffalo DNA.
步骤4)中所述的酶切，是将两种扩增产物5μL分别与双蒸水3.5μL、10×Buffer1μL、10U/μL的内切酶0.5μL混匀后离心，37℃培养箱放置2-4h。For the enzyme digestion described in step 4), 5 μL of the two amplification products were mixed with 3.5 μL of double distilled water, 1 μL of 10×Buffer, and 0.5 μL of endonuclease at 10 U/μL, and then centrifuged, and placed in a 37°C incubator for 2 -4h.
本发明的效果是：本发明可以快速、准确地鉴别杂交水牛染色体核型数，可以作为杂交水牛的分子标记选育手段，加速杂交水牛的选育效率。The effect of the invention is that the invention can quickly and accurately identify the karyotype number of hybrid buffalo chromosomes, can be used as a molecular marker breeding method for hybrid buffaloes, and accelerates the breeding efficiency of hybrid buffaloes.
更详细的技术方案见《具体实施方式》。For more detailed technical solutions, see "Specific Implementation Modes".
附图说明Description of drawings
图1：是本发明的技术流程示意图。Fig. 1: is the technical flow diagram of the present invention.
图2：是水牛RBP3基因一个片段RBP3-1的DNA序列、突变位点及引物。Figure 2: DNA sequence, mutation site and primers of RBP3-1, a fragment of buffalo RBP3 gene.
图3：是水牛RBP3基因另一个片段RBP3-2的DNA序列、突变位点及引物。Figure 3: The DNA sequence, mutation site and primers of another segment of the buffalo RBP3 gene, RBP3-2.
图4：是RBP3-1的365bp处A365-T365的碱基突变。Figure 4: It is the base mutation of A365-T365 at 365bp of RBP3-1.
图5：是三种核型的RBP3-1酶切电泳图谱。Figure 5: is the three karyotypes of RBP3-1 enzyme digestion electrophoresis patterns.
图6：是RBP3-2的442bp处G442-A442的碱基突变。Figure 6: The base mutation of G442-A442 at 442bp of RBP3-2.
图7：是三种核型的RBP3-2酶切电泳图谱。Figure 7: is the RBP3-2 restriction electrophoresis pattern of three karyotypes.
图8：是2N＝48杂交水牛染色体核型。Figure 8: is the 2N=48 hybrid buffalo chromosome karyotype.
图9：是2N＝49杂交水牛染色体核型。Figure 9: is the 2N=49 hybrid buffalo chromosome karyotype.
图10：是2N＝50杂交水牛染色体核型。Figure 10: is the 2N=50 hybrid buffalo chromosome karyotype.
具体实施方式detailed description
实施例1Example 1
(一)RBP3基因两段DNA序列PCR扩增(1) PCR amplification of two DNA sequences of RBP3 gene
1.引物设计1. Primer Design
用黄牛RBP3基因作模板序列，设计引物对，根据SEQIDNO：1所述RBP3-1的正向引物为5’-GTGCCGCTGCTACTCTCTTACT-3’，反向引物为5’-TCCTTACCTATGTGACGCTTGACG-3；根据SEQIDNO：2所述RBP3-2的正向引物为5’-AACACCTATCCACGACCTTTATCAT-3’，反向引物为5’-AGTTGCTTTAGCGAAGACACTATTATC-3’。用普通PCR方法扩增杂交水牛RBP3基因的两段DNA片段，PCR产物纯化和测序，通过序列分析获得如序列表SEQIDNO：1和SEQIDNO：2所述的序列；在RBP3-1的365bp处有一个A365-T365的碱基突变；在RBP3-2的442bp处有一个G442-A442的碱基突变。可辨别杂交水牛的不同核型。Using the cattle RBP3 gene as a template sequence, design a pair of primers. According to SEQ ID NO: 1, the forward primer of RBP3-1 is 5'-GTGCCGCTGCTACTCTCTTACT-3', and the reverse primer is 5'-TCCTTACCTATGTGACGCTTGACG-3; according to SEQ ID NO: 2 The forward primer of RBP3-2 is 5'-AACACCTATCCACGACCTTTATCAT-3', and the reverse primer is 5'-AGTTGCTTTAGCGAAGACACTATTATC-3'. Amplify two DNA fragments of hybrid buffalo RBP3 gene by ordinary PCR method, purify and sequence the PCR product, and obtain the sequence as described in the sequence table SEQIDNO: 1 and SEQIDNO: 2 through sequence analysis; there is a There is a base mutation of A365-T365; there is a base mutation of G442-A442 at 442bp of RBP3-2. Different karyotypes of hybrid buffaloes can be distinguished.
2.PCR产物的纯化和测序2. Purification and sequencing of PCR products
PCR扩增：PCR amplification:
1)SEQIDNO:11) SEQ ID NO: 1
20μL体系：双蒸水8μL、Mix10μL、正向引物0.5μL、反向引物0.5μL、杂交水牛DNA1μL。扩增条件：94℃预变性5min，94℃变性40s，55.2℃退火40s，72℃延伸40s，35个循环，72℃再延伸5min，16℃保持5min。共51个样本，PCR产物经2％琼脂糖凝胶电泳检测。20 μL system: 8 μL of double distilled water, 10 μL of Mix, 0.5 μL of forward primer, 0.5 μL of reverse primer, 1 μL of hybrid buffalo DNA. Amplification conditions: 94°C pre-denaturation for 5 min, 94°C denaturation for 40 s, 55.2°C annealing for 40 s, 72°C extension for 40 s, 35 cycles, 72°C extension for 5 min, 16°C for 5 min. A total of 51 samples, PCR products were detected by 2% agarose gel electrophoresis.
2)SEQIDNO:22) SEQ ID NO: 2
20μL体系：双蒸水8μL、Mix10μL、正向引物0.5μL、反向引物0.5μL、杂交水牛DNA1μL。扩增条件：94℃预变性5min，94℃变性30s，52℃退火30s，72℃延伸30s，35个循环，72℃再延伸5min，16℃保持5min。共51个样本，PCR产物经2％琼脂糖凝胶电泳检测。送公司测序。20 μL system: 8 μL of double distilled water, 10 μL of Mix, 0.5 μL of forward primer, 0.5 μL of reverse primer, 1 μL of hybrid buffalo DNA. Amplification conditions: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, 35 cycles, extension at 72°C for 5 min, and holding at 16°C for 5 min. A total of 51 samples, PCR products were detected by 2% agarose gel electrophoresis. Sent to the company for sequencing.
(二)PCR-RFLP诊断方法建立(2) Establishment of PCR-RFLP diagnostic method
RFLP检测：RFLP detection:
1)SEQIDNO:11) SEQ ID NO: 1
10μL酶切体系：双蒸水3.5μL、10×Buffer1μL、限制性内切酶Ecil0.5μL(10U/μL)、PCR产物5μL。将样品混匀后离心，37℃培养箱放置2-4h。2％琼脂糖凝胶电泳检测酶切结果。10 μL digestion system: 3.5 μL of double distilled water, 1 μL of 10×Buffer, 0.5 μL of restriction endonuclease Ecil (10 U/μL), 5 μL of PCR product. The samples were mixed and centrifuged, and placed in a 37°C incubator for 2-4h. 2% agarose gel electrophoresis to detect enzyme digestion results.
扩增杂交水牛基因组DNA得到了1008bp左右大小的特异性扩增片段，序列分析结果表明在365bp处存在A365-T365突变。该基因突变位点由两个等位基因控制，其中T是没有形成酶切位点的等位基因，A是形成酶切位点的等位基因。这两个等位基因可组成三种基因型，其中TT型为未发生酶切的纯合型(电泳检测时只有1008bp一条DNA带)，AA型为发生酶切的纯合型(电泳检测时出现642bp和365bp两条DNA带)，TC为杂合型(电泳检测时出现1008bp、642bp和365bp三条DNA带)。The amplified hybrid buffalo genomic DNA obtained a specific amplified fragment with a size of about 1008bp, and the sequence analysis results showed that there was an A365-T365 mutation at 365bp. The gene mutation site is controlled by two alleles, wherein T is the allele that does not form a restriction site, and A is the allele that forms a restriction site. These two alleles can form three genotypes, among which the TT type is the homozygous type without enzyme digestion (there is only one DNA band of 1008bp when detected by electrophoresis), and the AA type is the homozygous type with enzyme digestion (when detected by electrophoresis). Two DNA bands of 642bp and 365bp appeared), and TC was heterozygous (three DNA bands of 1008bp, 642bp and 365bp appeared in electrophoresis detection).
2)SEQIDNO:22) SEQ ID NO: 2
10μL酶切体系：双蒸水3.5μL、10×Buffer1μL、限制性内切酶Drdl0.5μL(10U/μL)、PCR产物5μL。将样品混匀后离心，37℃培养箱放置2-4h。2％琼脂糖凝胶电泳检测酶切结果。10 μL digestion system: 3.5 μL of double distilled water, 1 μL of 10×Buffer, 0.5 μL of restriction enzyme Drdl (10 U/μL), 5 μL of PCR product. The samples were mixed and centrifuged, and placed in a 37°C incubator for 2-4h. 2% agarose gel electrophoresis to detect enzyme digestion results.
扩增杂交水牛基因组DNA得到了747bp左右大小的特异性扩增片段，序列分析结果表明在442bp处存在G442-A442突变.该基因突变位点由两个等位基因控制，其中A是没有形成酶切位点的等位基因，G是形成酶切位点的等位基因。这两个等位基因可组成三种基因型，其中AA型为未发生酶切的纯合型(电泳检测时只有747bp一条DNA带)，GG型为发生酶切的纯合型(电泳检测时出442bp和305bp两条DNA带)，AG为杂合型(电泳检测时出现747bp、442bp和305bp三条DNA带)。Amplified hybrid buffalo genomic DNA obtained a specific amplified fragment with a size of about 747bp, and the sequence analysis results showed that there was a G442-A442 mutation at 442bp. The gene mutation site was controlled by two alleles, of which A was no enzyme G is the allele for the cleavage site, and G is the allele for the restriction site. These two alleles can form three genotypes, of which the AA type is the homozygous type without enzyme digestion (only a DNA band of 747 bp is detected by electrophoresis), and the GG type is the homozygous type with enzyme digestion (the DNA band is only 747 bp when detected by electrophoresis). There were two DNA bands of 442bp and 305bp), and AG was heterozygous (three DNA bands of 747bp, 442bp and 305bp were detected by electrophoresis).
水牛RBP3基因的两个突变位点可以辨别杂交水牛的核型，对51头杂交水牛进行酶切分型，核型数由传统的核型分析方法所得，得到的数据如下：The two mutation sites of buffalo RBP3 gene can distinguish the karyotype of hybrid buffalo, and 51 hybrid buffalo were typed by enzyme digestion. The number of karyotypes was obtained by the traditional karyotype analysis method, and the obtained data are as follows:
表1不同核型的酶切条带统计(个体数)Table 1 Statistics of enzyme-digested bands of different karyotypes (number of individuals)
*表示差异显著，**表示差异极显著* indicates significant difference,** indicates extremely significant difference
不同核型的酶切条带差异比较大，2个酶切位点的卡方检验均极显著，说明不同核型之间的酶切条带是显著不同的，酶切条带是可以作为辨别不同核型的依据。The difference between the restriction bands of different karyotypes is relatively large, and the chi-square test of the two restriction sites is extremely significant, indicating that the restriction bands between different karyotypes are significantly different, and the restriction bands can be used as a discrimination Basis for different karyotypes.
RBP3-1的酶切条带中，核型数为48的杂交水牛没有被酶切，但是1条酶切条带中2n＝48核型得比例只占52.94％；2条酶切条带中2n＝50核型比例达90.91％，但无法做到100％准确。In the enzyme-digested band of RBP3-1, the hybrid buffalo with a karyotype of 48 was not digested, but the proportion of karyotype 2n=48 in one enzyme-digested band only accounted for 52.94%; in the two enzyme-digested bands The karyotype ratio of 2n=50 reaches 90.91%, but it cannot be 100% accurate.
RBP3-2的酶切条带中，只有核型数为48的杂交水牛没有被酶切，1条酶切条带全为2n＝48核型，比例100％；2条酶切条带中2n＝48核型比例65％；3条酶切条带全为2n＝49核型，比例100％。因此，酶切条带为1条和3条的个体可以确定为核型数为2n＝48和2n＝49的杂交水牛。Among the enzyme-digested bands of RBP3-2, only the hybrid buffalo with a karyotype of 48 was not digested, and one enzyme-digested band was all 2n=48 karyotypes, and the ratio was 100%; 2n in the two enzyme-digested bands The proportion of =48 karyotype is 65%; the three restriction bands are all 2n=49 karyotype, and the proportion is 100%. Therefore, individuals with 1 and 3 restriction bands can be determined as hybrid buffaloes with karyotype numbers 2n=48 and 2n=49.
综上，可依据RBP3-1的酶切条带(条带为2的是2n＝50核型)辨别2n＝50核型的水牛，依据RBP3-2的酶切条带辨别核型2n＝48的杂交水牛(条带为1)，2n＝49核型的杂交水牛(条带为3)，从而辨别三种核型。To sum up, the buffalo with 2n=50 karyotype can be distinguished according to the enzyme-digested band of RBP3-1 (the band with 2 is 2n=50 karyotype), and the karyotype 2n=48 can be distinguished according to the enzyme-digested band of RBP3-2 The hybrid buffalo (the band is 1), the hybrid buffalo with 2n=49 karyotypes (the band is 3), so as to distinguish the three karyotypes.
| Application Number | Priority Date | Filing Date | Title |
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| CN201410332529.3ACN104152549B (en) | 2014-07-14 | 2014-07-14 | A kind of method of quick discriminating hybridization buffalo karyotype |
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| CN201410332529.3ACN104152549B (en) | 2014-07-14 | 2014-07-14 | A kind of method of quick discriminating hybridization buffalo karyotype |
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| CN119736416A (en)* | 2025-03-06 | 2025-04-01 | 吉林大学 | A method for rapid identification of chromosome karyotypes of hybrid deer |
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