中国水稻科学 ›› 2021, Vol. 35 ›› Issue (3): 303-310.DOI: 10.16819/j.1001-7216.2021.0717
• 研究报告 • 上一篇
刘树芳1,2,#, 董丽英1,2,#, 李迅东1,2, 周伍民1,3, 杨勤忠1,2,*()
收稿日期:
2020-07-24
修回日期:
2020-11-24
出版日期:
2021-05-10
发布日期:
2021-05-10
通讯作者:
杨勤忠
作者简介:
#共同第一作者;
基金资助:
Shufang LIU1,2,#, Liying DONG1,2,#, Xundong LI1,2, Wumin ZHOU1,3, Qinzhong YANG1,2,*()
Received:
2020-07-24
Revised:
2020-11-24
Online:
2021-05-10
Published:
2021-05-10
Contact:
Qinzhong YANG
About author:
#These authors contributed equally to this work;
摘要:
【目的】Pi9是一个广谱稻瘟病抗性基因,田间病圃监测发现持有Pi9的水稻单基因系IRBL9-W在苗期高抗稻瘟病,但却感穗瘟。探明水稻单基因系IRBL9-W在苗期抗病而孕穗末期感染穗颈瘟的原因,为Pi9基因在水稻抗病育种中的有效利用提供参考。【方法】利用从IRBL9-W穗颈瘟病斑上分离的8个单孢菌株以及实验室保存的单孢菌株Y363,在温室分别对单基因系IRBL9-W苗期和孕穗末期进行接种鉴定;并利用病原菌AvrPi9基因的特异引物对9个单孢菌株进行PCR扩增及产物测序;提取水稻单基因系IRBL9-W苗期叶片和抽穗期穗部的总RNA,通过半定量RT-PCR以及实时qRT-PCR分析Pi9基因在苗期和穗期的表达。【结果】在温室人工喷雾接种条件下,IRBL9-W在苗期对从穗颈瘟上分离的8个单孢及对照菌株Y363均表现为抗病;随机选取的2个从IRBL9-W穗颈瘟病样分离的单孢菌株(YX2-7-1和YX2-15-1)及对照菌株Y363对孕穗末期IRBL9-W注射接种,接种的植株表现出典型的穗颈瘟症状;AvrPi9的等位基因分析结果表明,与AvrPi9相比,Y363中的等位基因与AvrPi9完全相同,而从IRBL9-W穗瘟分离的8个单孢菌株中编码区与AvrPi9基因完全相同,但在编码起始位置上游-264 bp处缺失16 bp的一段序列。由于IRBL9-W苗期对这些菌株均表现抗病,推测这段序列的缺失并不影响AvrPi9基因的功能;实时qRT-PCR分析结果表明,Pi9基因在穗部的表达量为苗期叶片表达量的47.3%。【结论】在水稻单基因系IRBL9-W中,与苗期叶片中Pi9基因的表达量相比,Pi9基因在穗部表达量的明显降低可能是造成IRBL9-W穗期感稻瘟病的原因。
刘树芳, 董丽英, 李迅东, 周伍民, 杨勤忠. 持有Pi9基因的水稻单基因系IRBL9-W对稻瘟病菌苗期和成株期抗性差异[J]. 中国水稻科学, 2021, 35(3): 303-310.
Shufang LIU, Liying DONG, Xundong LI, Wumin ZHOU, Qinzhong YANG. Different Reactions of Rice Monogenic Line IRBL9-W Harboring Pi9 Gene to Magnaporthe oryzae Containing AvrPi9 During Seedling and Adult-plant Stages[J]. Chinese Journal OF Rice Science, 2021, 35(3): 303-310.
单基因系 Monogenic line | 抗性基因 R gene | 稻瘟病菌株 Magnaporthe oryzae strains | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
YX2-2-1 | YX2-3-2 | YX2-5-1 | YX2-6-1 | YX2-10-1 | YX2-14-1 | YX2-7-1 | YX2-15-1 | Y363 | ||
IRBLA-A | Pia | R | R | R | R | R | R | S | S | S |
IRBLI-F5 | Pii | R | S | R | S | S | R | S | S | S |
IRBLKS-F5 | Piks | S | S | S | S | S | S | S | S | S |
KRBLK KA | Pik | S | S | S | S | S | S | S | S | S |
KRBLKP-K60 | Pikp | S | S | S | S | S | S | S | S | R |
KRBLKH-K3 | Pikh | R | R | R | R | R | R | R | S | R |
IRBLZ5-CA | Pi2 | S | S | S | S | S | S | S | S | S |
IRBLZ FU | Piz | S | S | S | S | S | S | S | S | S |
IRBLZT-T | Pizt | R | R | R | R | R | R | R | R | R |
IRBLTA-K1 | Pita | S | S | S | S | S | S | S | S | S |
IRBLB-B | Pib | S | S | S | S | S | S | S | S | S |
IRBLT-K59 | Pit | R | R | R | R | R | R | S | S | S |
IRBLSH-B | Pish | S | S | S | S | S | S | S | S | R |
IRBL1-CL | Pi1 | R | R | R | R | R | R | R | S | S |
IRBL3-CP4 | Pi3 | S | S | S | S | S | S | S | S | S |
IRBL5-M | Pi5 | R | R | R | R | R | R | R | R | R |
IRBL7-M | Pi7 | S | S | S | S | R | S | S | S | R |
IRBL12-M | Pi12 | S | S | S | S | S | S | S | S | R |
IRBL19-A | Pi19 | S | S | S | S | R | S | S | S | S |
IRBLKM-TS | Pikm | R | R | R | R | R | R | R | S | S |
IRBL20-IR24 | Pi20 | R | R | R | R | R | R | R | R | R |
IRBLTA2-PI | Pita2 | S | S | S | S | S | S | R | S | R |
IRBL11-ZH | Pi11 | S | S | S | S | S | S | S | S | S |
IRBL9-W | Pi9 | R | R | R | R | R | R | R | R | R |
LTH | - | S | S | S | S | S | S | S | S | S |
表1 稻瘟病菌单孢菌株在单基因系上的致病性测定
Table 1 Pathotyping of Magnaporthe oryzae strains on monogenic lines.
单基因系 Monogenic line | 抗性基因 R gene | 稻瘟病菌株 Magnaporthe oryzae strains | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
YX2-2-1 | YX2-3-2 | YX2-5-1 | YX2-6-1 | YX2-10-1 | YX2-14-1 | YX2-7-1 | YX2-15-1 | Y363 | ||
IRBLA-A | Pia | R | R | R | R | R | R | S | S | S |
IRBLI-F5 | Pii | R | S | R | S | S | R | S | S | S |
IRBLKS-F5 | Piks | S | S | S | S | S | S | S | S | S |
KRBLK KA | Pik | S | S | S | S | S | S | S | S | S |
KRBLKP-K60 | Pikp | S | S | S | S | S | S | S | S | R |
KRBLKH-K3 | Pikh | R | R | R | R | R | R | R | S | R |
IRBLZ5-CA | Pi2 | S | S | S | S | S | S | S | S | S |
IRBLZ FU | Piz | S | S | S | S | S | S | S | S | S |
IRBLZT-T | Pizt | R | R | R | R | R | R | R | R | R |
IRBLTA-K1 | Pita | S | S | S | S | S | S | S | S | S |
IRBLB-B | Pib | S | S | S | S | S | S | S | S | S |
IRBLT-K59 | Pit | R | R | R | R | R | R | S | S | S |
IRBLSH-B | Pish | S | S | S | S | S | S | S | S | R |
IRBL1-CL | Pi1 | R | R | R | R | R | R | R | S | S |
IRBL3-CP4 | Pi3 | S | S | S | S | S | S | S | S | S |
IRBL5-M | Pi5 | R | R | R | R | R | R | R | R | R |
IRBL7-M | Pi7 | S | S | S | S | R | S | S | S | R |
IRBL12-M | Pi12 | S | S | S | S | S | S | S | S | R |
IRBL19-A | Pi19 | S | S | S | S | R | S | S | S | S |
IRBLKM-TS | Pikm | R | R | R | R | R | R | R | S | S |
IRBL20-IR24 | Pi20 | R | R | R | R | R | R | R | R | R |
IRBLTA2-PI | Pita2 | S | S | S | S | S | S | R | S | R |
IRBL11-ZH | Pi11 | S | S | S | S | S | S | S | S | S |
IRBL9-W | Pi9 | R | R | R | R | R | R | R | R | R |
LTH | - | S | S | S | S | S | S | S | S | S |
图2 不同菌株中AvrPi9的扩增分析 M–DNA分子量标记; 条带1~9分别指稻瘟病菌菌株YX2-2-1, YX2-3-2, YX2-5-1, YX2-6-1, YX2-10-1, YX2-14-1, YX2-7-1, YX2-15-1和Y363。
Fig. 2. Amplification of AvrPi9 from different M. oryzae strains. M, DNA molecular marker; DL2000; Lane 1 to Lane 9 refer to YX2-2-1, YX2-3-2, YX2-5-1, YX2-6-1, YX2-10-1, YX2-14-1, YX2-7-1, YX2-15-1 and Y363, respectively.
稻瘟病菌株 M. oryzae strain | 发病级别 Disease scores | |||||
---|---|---|---|---|---|---|
0 | 1 | 3 | 5 | 7 | 9 | |
YX2-7-1 | 0a | 0 | 0 | 1 | 1 | 13 |
YX2-15-1 | 0 | 0 | 0 | 0 | 1 | 13 |
Y363 | 0 | 0 | 0 | 0 | 1 | 14 |
空白对照CKb | 15 | 0 | 0 | 0 | 0 | 0 |
表2 孕穗末期利用3个稻瘟病菌菌株接种IRBL9-W后的穗瘟调查结果
Table 2 Investigating results of panicle blast of IRBL9-W inoculated with three M. oryzae strains at late-booting stage.
稻瘟病菌株 M. oryzae strain | 发病级别 Disease scores | |||||
---|---|---|---|---|---|---|
0 | 1 | 3 | 5 | 7 | 9 | |
YX2-7-1 | 0a | 0 | 0 | 1 | 1 | 13 |
YX2-15-1 | 0 | 0 | 0 | 0 | 1 | 13 |
Y363 | 0 | 0 | 0 | 0 | 1 | 14 |
空白对照CKb | 15 | 0 | 0 | 0 | 0 | 0 |
图1 单基因系IRBL9-W孕穗末期及苗期接种的症状 A~C–IRBL9-W接种菌株YX2-7-1、YX2-15-1和Y363在孕穗末期的症状; D~F–IRBL9-W(左)和丽江新团黑谷(右)苗期接种菌株YX2-7-1、YX2-15-1和Y363后的症状。
Fig. 1. Symptoms of monogenic line IRBL9-W inoculated with M. oryzae fungus at late-booting stage and seedling stage. A–C, Symptoms of monogenic line IRBL9-W inoculated with M. oryze strains YX2-7-1, YX2-15-1 and Y363 at late booting stage, respectively. D–F: Symptoms of monogenic line IRBL9-W (left) and Lijiangxintuanheigu (right) inoculated with M. oryzae strains YX2-7-1, YX2-15-1 and Y363 at seedling stage, respectively.
图3 Pi9基因的表达分析 A–Pi9基因的半定量分析。从成株期穗部(P)及苗期叶片(L)制备的cDNA用作PCR模板进行扩增,Actin1 用作内参基因;B–实时荧光定量RT-PCR比较分析Pi9基因在苗期和穗期的表达量。
Fig. 3. Expression level of Pi9 gene. A, Expression analysis of Pi9 gene by semi-quantitative RT-PCR method. The cDNA prepared from panicles (P) of adult plants and seedling leaves (L) of IRBL9-W carrying Pi9 gene was used as template for PCR amplification, with Actin 1 as reference gene; B, Comparative analysis of relative expression level of Pi9 gene in panicle and seedling leaves of IRBL9-W by real-time quantitative RT-PCR.
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