中国白梨15个主栽品种S基因型及S_（29）-RNase基因的鉴定
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摘要
自交不亲和性是被子植物中常见的一种生物学现象，是植物在长期进化过程中形成的防止近亲繁殖和物种退化的一种保护机制。梨是我国重要的果树之一，属配子体自交不亲和类型。目前生产上主要采用配置授粉品种的方法来克服梨品种的自交不亲和性，以保证高产稳产。白梨是我国主要的梨栽培系统之一，国内外对白梨品种S基因及S基因型鉴定方面的研究还刚刚起步，到2003年止，白梨中分离鉴定了8个新的S基因，并鉴定了近20个品种的S基因型。本论文以白梨15个品种为材料对其自交不亲和基因及品种基因型进行了研究，其主要结果如下：
利用梨S_(1-8)-等位基因引物‘FTQQYQ’(P1)或‘5′-32bp’(P3)和‘IIWPNV’(P2)对白梨15个品种基因组DNA进行了S基因特异性PCR扩增，每个品种扩增出1-2个特异性DNA片段。通过PCR-RFLP检测方法和DNA片段的回收测序，及生物信息学分析，确定了11个品种的S基因型和4个品种一个S等位基因。大核白为S_(16)S_(19)，马蹄黄为S_(16)S_(19)，大水核为S_7S_(19)，油梨为S_(16)S_(19)，大理鸡腿为S_(17)S_(19)，硬枝青为S_(12)S_(12)，恩梨为S_1S_(19)，天生伏为S_(29)S_(12)，锦丰为S_(16)S_8，冬黄为S_(20)S_8，大鸭梨为S_1S_1S_(21)S_(21)，蜜梨为S_(29)S_x，紫酥为S_(19)S_x，耀县银梨为S_(21)S_x，秋白为S_(19)S_x。
利用DNA片段回收测序方法，从天生伏和蜜梨中分离鉴定了一个新的S基因，命名为S_(29)等位基因，在GeneBank中的接受号为AY601098。P1P2间DNA片段与沙梨S_(1-16)等位基因以及白梨中新发现的S等位基因具有很高的同源性，其相似性达79％～99％，其推导氨基酸序列与S_(1-28)等位基因的相似性达62-97％。此新S等位基因的P3+信号肽+P1+C1+C2+HV+内含子+P2区域的DNA序列大小为472bp，其中，内含子大小为147bp，其推导氨基酸序列大小为99，包括了信号肽、C1区、C2区、与S-RNase识别相关的HV区、与S-RNase结构有关的3个半胱氨酸残基以及与RNase功能有关的1个组氨酸残基。信号肽由27个氨基酸组成，C1区为11个氨基酸，C2区为10个氨基酸，HV区为15个氨基酸。
总之，这些品种S基因型的确定及新S等位基因的发现为白梨授粉品种的配置及果树自交不亲和的深入研究提供科学依据。
Self-incompatibility, a common biological phenomenon in angiosperm, is an important protective mechanism of preventing close breeding and species retrogression that formed during long period evolution. Pear, an important fruit tree in China, belongs to the kind of gametophytic self-incompatibility (GS1). At present to arrange varieties for pollination or to perform artificial pollination is mainly adept to overcome SI for high and stable yield during the planting of pear. Brelschnider pear (Pyrus brelschnider) is a primary planting genealogy of pear in China. The researches on self-incompatible genes and S-genotypes of bretschnider pear are still in initial stage, 8 new S genes were isolated and determined and almost 20 cultivars' genotypes were determined in Bretschnider pear (Pyrus bretschnider) until 2003. In this paper, the researches on identification of S-genes and determination of S-genotypes of pear varieties are performed from 15 pear cultivars of bretschnider pear. The main results of this research are following as:
The PCR on genomic DNA of 15 varieties of bretschnider pear were performed using special primers 'FTQQYQ'(Pl) or '5 ' -32bp'(P3) and 'IIWPNV'(P2) of pear S1-8-RNase, 1-2 special DNA fragments were obtained from each variety. The genotype of 11 varieties and one S-allelic gene of 4 varieties of Bretschnider pear are determined by PCR-RFLP system, DNA sequencing, and bioinformatics analysis. The genotypes of Dahebai, Yinzhiqing, Enli, Tianshengfu, Jinfeng, Donghuang, Dayali, Mili, Zisu, Yaoxianyinli, and Qoubai are S16S19, S16S19, S7S19, S15S19, S17S19, S12S12,
S1S19, S29S12, S16S8, S20S8, S1S1S21S21, S29SX, S19SX, S2lSX, S19SX.
A new S-gene, named S29, is isolated and determined from two varieties (Mili and Tianshengfu) by sequencing. The GeneBank accession number is AY601098. The isolated DNA fragments between PI and P2 display high homology (similarity of 79-99 %) with those of S1-16-RNase.and new determined S-genes in bretschnider pear, and the similarity between it's putative amino sequence and S1-28-RNase is up to 62-97%. The DNA bp numbers of the sum of P3, signal peptide, P1, C2, C2, HV, introns, and P2 of the new S-gene is 472. The DNA bp numbers of introns of the new S-gene is 147. The lengths of putative amino acids of the gene fragment is 99, it contains signal peptide, C1 region, C2 region, HV region related to the recognition function of S-RNase, 3 cysteine residues related to the structure of S-RNase and 1 histidine residue related to the activity of RNase. The residue length of signal peptide, C1 region, C2 region, and HV region is 27, 11, 10 and 15 respectively. To summary, the determination of genotypes of these varieties and discovery of new S-RNase gene provide an important scientific foundation for arranging pollination varieties and further study of SI in fruit tree.
利用梨S_(1-8)-等位基因引物‘FTQQYQ’(P1)或‘5′-32bp’(P3)和‘IIWPNV’(P2)对白梨15个品种基因组DNA进行了S基因特异性PCR扩增，每个品种扩增出1-2个特异性DNA片段。通过PCR-RFLP检测方法和DNA片段的回收测序，及生物信息学分析，确定了11个品种的S基因型和4个品种一个S等位基因。大核白为S_(16)S_(19)，马蹄黄为S_(16)S_(19)，大水核为S_7S_(19)，油梨为S_(16)S_(19)，大理鸡腿为S_(17)S_(19)，硬枝青为S_(12)S_(12)，恩梨为S_1S_(19)，天生伏为S_(29)S_(12)，锦丰为S_(16)S_8，冬黄为S_(20)S_8，大鸭梨为S_1S_1S_(21)S_(21)，蜜梨为S_(29)S_x，紫酥为S_(19)S_x，耀县银梨为S_(21)S_x，秋白为S_(19)S_x。
利用DNA片段回收测序方法，从天生伏和蜜梨中分离鉴定了一个新的S基因，命名为S_(29)等位基因，在GeneBank中的接受号为AY601098。P1P2间DNA片段与沙梨S_(1-16)等位基因以及白梨中新发现的S等位基因具有很高的同源性，其相似性达79％～99％，其推导氨基酸序列与S_(1-28)等位基因的相似性达62-97％。此新S等位基因的P3+信号肽+P1+C1+C2+HV+内含子+P2区域的DNA序列大小为472bp，其中，内含子大小为147bp，其推导氨基酸序列大小为99，包括了信号肽、C1区、C2区、与S-RNase识别相关的HV区、与S-RNase结构有关的3个半胱氨酸残基以及与RNase功能有关的1个组氨酸残基。信号肽由27个氨基酸组成，C1区为11个氨基酸，C2区为10个氨基酸，HV区为15个氨基酸。
总之，这些品种S基因型的确定及新S等位基因的发现为白梨授粉品种的配置及果树自交不亲和的深入研究提供科学依据。
Self-incompatibility, a common biological phenomenon in angiosperm, is an important protective mechanism of preventing close breeding and species retrogression that formed during long period evolution. Pear, an important fruit tree in China, belongs to the kind of gametophytic self-incompatibility (GS1). At present to arrange varieties for pollination or to perform artificial pollination is mainly adept to overcome SI for high and stable yield during the planting of pear. Brelschnider pear (Pyrus brelschnider) is a primary planting genealogy of pear in China. The researches on self-incompatible genes and S-genotypes of bretschnider pear are still in initial stage, 8 new S genes were isolated and determined and almost 20 cultivars' genotypes were determined in Bretschnider pear (Pyrus bretschnider) until 2003. In this paper, the researches on identification of S-genes and determination of S-genotypes of pear varieties are performed from 15 pear cultivars of bretschnider pear. The main results of this research are following as:
The PCR on genomic DNA of 15 varieties of bretschnider pear were performed using special primers 'FTQQYQ'(Pl) or '5 ' -32bp'(P3) and 'IIWPNV'(P2) of pear S1-8-RNase, 1-2 special DNA fragments were obtained from each variety. The genotype of 11 varieties and one S-allelic gene of 4 varieties of Bretschnider pear are determined by PCR-RFLP system, DNA sequencing, and bioinformatics analysis. The genotypes of Dahebai, Yinzhiqing, Enli, Tianshengfu, Jinfeng, Donghuang, Dayali, Mili, Zisu, Yaoxianyinli, and Qoubai are S16S19, S16S19, S7S19, S15S19, S17S19, S12S12,
S1S19, S29S12, S16S8, S20S8, S1S1S21S21, S29SX, S19SX, S2lSX, S19SX.
A new S-gene, named S29, is isolated and determined from two varieties (Mili and Tianshengfu) by sequencing. The GeneBank accession number is AY601098. The isolated DNA fragments between PI and P2 display high homology (similarity of 79-99 %) with those of S1-16-RNase.and new determined S-genes in bretschnider pear, and the similarity between it's putative amino sequence and S1-28-RNase is up to 62-97%. The DNA bp numbers of the sum of P3, signal peptide, P1, C2, C2, HV, introns, and P2 of the new S-gene is 472. The DNA bp numbers of introns of the new S-gene is 147. The lengths of putative amino acids of the gene fragment is 99, it contains signal peptide, C1 region, C2 region, HV region related to the recognition function of S-RNase, 3 cysteine residues related to the structure of S-RNase and 1 histidine residue related to the activity of RNase. The residue length of signal peptide, C1 region, C2 region, and HV region is 27, 11, 10 and 15 respectively. To summary, the determination of genotypes of these varieties and discovery of new S-RNase gene provide an important scientific foundation for arranging pollination varieties and further study of SI in fruit tree.
引文
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