园艺学报 ›› 2022, Vol. 49 ›› Issue (1): 157-165.doi: 10.16420/j.issn.0513-353x.2020-0758
李茂福1,2,3, 杨媛1,2,4, 王华1,2,3, 范又维1,2,3, 孙佩1,2,3, 金万梅1,2,3,*()
收稿日期:
2021-03-30
修回日期:
2021-09-07
出版日期:
2022-01-25
发布日期:
2022-01-24
通讯作者:
金万梅
E-mail:jwm0809@163.com
基金资助:
LI Maofu1,2,3, YANG Yuan1,2,4, WANG Hua1,2,3, FAN Youwei1,2,3, SUN Pei1,2,3, JIN Wanmei1,2,3,*()
Received:
2021-03-30
Revised:
2021-09-07
Online:
2022-01-25
Published:
2022-01-24
Contact:
JIN Wanmei
E-mail:jwm0809@163.com
摘要:
以月季‘月月红’(Rosa chinensis‘Slater’s Crimson China’)和‘2015-58-20’(Rosa sp.)为材料进行自交和异交,授粉花柱中花粉管荧光显微镜观察结果显示月季为典型的配子体自交不亲和植物。通过与已知蔷薇科自交不亲和S-RNase基因的序列进行比对,在‘月月红’中克隆到RcS1-RNase和RcS2-RNase基因的cDNA和DNA全长序列,序列分析表明,RcS1-RNase和RcS2-RNase具有5个保守结构域(C1、C2、C3、RC4和C5)和1个高变区。系统进化分析发现,RcS-RNase与其他蔷薇科植物的S-RNase蛋白具有较高的同源性。组织特异性表达分析表明,RcS1-RNase和RcS2-RNase基因只在花柱中特异表达。
中图分类号:
李茂福, 杨媛, 王华, 范又维, 孙佩, 金万梅. 月季自交不亲和性S-RNase的鉴定与分析[J]. 园艺学报, 2022, 49(1): 157-165.
LI Maofu, YANG Yuan, WANG Hua, FAN Youwei, SUN Pei, JIN Wanmei. Identification and Analysis of Self Incompatibility S-RNase in Rose[J]. Acta Horticulturae Sinica, 2022, 49(1): 157-165.
目的 Purpose | 基因 Gene | 引物序列(5′-3′) Primer sequence |
---|---|---|
基因克隆 Gene cloning | RcS1-RNase | F:ATGGGTTCCTATGAATATTTTAAATTTGTGG;R:CTACAGAATATCGATTGGAAAAGTATCTTGAC |
RcS2-RNase | F:ATGGCACAAAATAATATGGCTTACGTCCTC;R:TCAGTTCGGATATTCTATAAGAGTTGGAGC | |
RT-PCR | RcS1-RNase | F:GCGTTTAGTCTGCAATGACGCAGAAGCTC;R:ACTAGTATCAAACGAAATGATTGCTCGCG |
RcS2-RNase | F:TGTGGTAACAGCTGCAAAGC;R:TCAACCACGTTTTTGCCATA | |
Actin | F:GGCTGTTCTCTCTCTGTATGC;R:TTCTGGGCACCTGAATCTC |
表1 本研究中所用引物序列
Table 1 Primers sequence used in this study
目的 Purpose | 基因 Gene | 引物序列(5′-3′) Primer sequence |
---|---|---|
基因克隆 Gene cloning | RcS1-RNase | F:ATGGGTTCCTATGAATATTTTAAATTTGTGG;R:CTACAGAATATCGATTGGAAAAGTATCTTGAC |
RcS2-RNase | F:ATGGCACAAAATAATATGGCTTACGTCCTC;R:TCAGTTCGGATATTCTATAAGAGTTGGAGC | |
RT-PCR | RcS1-RNase | F:GCGTTTAGTCTGCAATGACGCAGAAGCTC;R:ACTAGTATCAAACGAAATGATTGCTCGCG |
RcS2-RNase | F:TGTGGTAACAGCTGCAAAGC;R:TCAACCACGTTTTTGCCATA | |
Actin | F:GGCTGTTCTCTCTCTGTATGC;R:TTCTGGGCACCTGAATCTC |
授粉 Pollination | 材料 Material | 授粉花朵数 Number of pollinated flowers | 收获果实数 Number of fruits | 坐果率/% Fruit-set ration | 种子数 Number of seeds |
---|---|---|---|---|---|
自花授粉 Self-pollination | 2015-58-20(P1) | 20 | 0 | 0 | 0 |
月月红(P2) Slater’s Crimson China | 51 | 0 | 0 | 0 | |
异花授粉 Cross-pollination | P1 × P2 | 235 | 96 | 40.86 | 362 |
表2 ‘月月红’与‘2015-58-20’授粉坐果率
Table 2 Comparison of fruit setting among four crosses
授粉 Pollination | 材料 Material | 授粉花朵数 Number of pollinated flowers | 收获果实数 Number of fruits | 坐果率/% Fruit-set ration | 种子数 Number of seeds |
---|---|---|---|---|---|
自花授粉 Self-pollination | 2015-58-20(P1) | 20 | 0 | 0 | 0 |
月月红(P2) Slater’s Crimson China | 51 | 0 | 0 | 0 | |
异花授粉 Cross-pollination | P1 × P2 | 235 | 96 | 40.86 | 362 |
图2 月季RcS1-RNase和RcS2-RNase基因克隆 RcS1-RNase和RcS2-RNase基因克隆
Fig. 2 Clone the RcS1-RNase and RcS2-RNase gene in rosa RcS1-RNase and RcS2-RNase gene in rosa
蛋白 Protein | 长度/aa Length | 分子量/kD MW | 等电点 pI | 天冬氨酸 + 谷氨酸 Asp(-)+ Glu(-) | 精氨酸 + 赖氨酸 Arg(+)+ Lys(+) | 不稳定性系数 Instablility index | 脂肪族氨基酸系数 Aliphatic aa index | 亲疏水性 Hydropathicity |
---|---|---|---|---|---|---|---|---|
RcS1-RNase | 206 | 24.04 | 8.74 | 20 | 25 | 37.78 | 67.23 | -0.66 |
RcS2-RNase | 219 | 24.84 | 7.57 | 19 | 20 | 35.73 | 71.28 | -0.30 |
表3 月季RcS1-RNase和RcS2-RNase的蛋白理化特性
Table 3 Physical and chemical property of RcS1-RNase and RcS2-RNase from Rosa chinensis
蛋白 Protein | 长度/aa Length | 分子量/kD MW | 等电点 pI | 天冬氨酸 + 谷氨酸 Asp(-)+ Glu(-) | 精氨酸 + 赖氨酸 Arg(+)+ Lys(+) | 不稳定性系数 Instablility index | 脂肪族氨基酸系数 Aliphatic aa index | 亲疏水性 Hydropathicity |
---|---|---|---|---|---|---|---|---|
RcS1-RNase | 206 | 24.04 | 8.74 | 20 | 25 | 37.78 | 67.23 | -0.66 |
RcS2-RNase | 219 | 24.84 | 7.57 | 19 | 20 | 35.73 | 71.28 | -0.30 |
图3 月季RcS1-RNase和RcS2-RNase与梨PbS21-RNase和苹果MdS34-RNase的保守区比对
Fig. 3 Alignment of the conserved amino acid region between the RcS1-RNase,RcS2-RNase and PbS21-RNase(Pyrus × bretschneideri),MdS34-RNase(Malus × domestica)Pyrus × bretschneideri),MdS34-RNase(Malus × domestica)
图4 月季RcS-RNase与其他植物S-RNase蛋白序列系统进化树 Pb:梨;Md:苹果;Pm:杏;Pa:樱桃;Ah:金鱼草;Na:烟草;Ph:矮牵牛;At:拟南芥。
Fig. 4 Phylogenetic tree of amino acid sequence of Rosa chinensis RcS-RNase and S-RNase of other plants Pb:Pyrus × bretschneideri;Md:Malus × domestica;Pm:Prunus mume;Pa:Prunus avium;Ah:Antirrhinum hispanicum;Na:Nicotiana alata;Ph:Petunia hybrid;At:Arabidopsis thaliana.
图5 月季RcS1-RNase和RcS2-RNase 组织特异性分析RcS1-RNase和RcS2-RNase 组织特异性分析
Fig. 5 The tissue specificity expression analysis of RcS1-RNase and RcS2-RNase in Rosa
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