梅花花瓣瞬时转化体系构建

doi:10.16420/j.issn.0513-353x.2024-0448

摘要/Abstract

摘要：

以梅花（Prunus mume）花瓣为受体，β-葡萄糖苷酸酶基因（GUS）为报告基因，通过单因素、正交试验对影响梅花花瓣瞬时转化效率的7个主要因素进行了分析。对不同侵染方式、侵染时间、发育阶段和基因型进行单因素试验。结果表明，整花真空法、侵染时间为15 min、发育阶段为初开期和‘雪梅’基因型的GUS瞬时表达率最高。三因素水平正交试验结果表明，对梅花花瓣GUS瞬时表达的影响大小依次为共培养时间、农杆菌类型、侵染浓度（OD₆₀₀值），其中，以农杆菌EHA105、OD₆₀₀值为0.6、共培养5 d的花瓣GUS瞬时表达率最高（32.67%），并以梅花花青素合成基因（PmANS）为目的基因对筛选出的最优组合进行了成功验证。

关键词: 梅花, 花瓣, 农杆菌介导法, 瞬时转化, GUS

Abstract:

This study takes the petals of Prunus mume as the receptor and GUS gene as the reporter gene. The influences of 7 main factors on the transient transformation efficiency of Prunus mume petals were conducted through an orthogonal design and single factor analysis method. The effects of infection mode，infection time，development stage and genotype on the transient transformation efficiency of Prunus mume petals were investigated by single factor experiment. The results showed that the whole flower vacuum method，infestation time of 15 min，development stage of initial blooming，and‘XueMei’ genotype had the highest transient expression rate of GUS gene，respectively. The orthogonal experiment was further designed to explore the effect of the combination of Agrobacterium strain type，OD₆₀₀ value，Coculture time on the transient transformation efficiency of Prunus mume petals. The results showed that the three factors affected GUS transient expression in the order of coculture time，Agrobacterium strains type，OD₆₀₀ value. Among them，the combination of Agrobacterium EHA105，OD₆₀₀values is 0.6 and coculture time is 5 d showed the highest GUS transient expression effect of Prunus mume petals（32.67%）. Then，the optimal combination screened was verified by applying an anthocyanin synthesis gene（PmANS）as the target gene.

Key words: Prunus mume, petal, Agrobacterium-mediated method, transient transformation, GUS

田壮, 房晨曦, 刘雅婷, 王雨桐, 薛林蕾, 石嘉欣, 任天放, 张俊卫, 包满珠, 张杰. 梅花花瓣瞬时转化体系构建[J]. 园艺学报, 2025, 52(4): 908-920.

TIAN Zhuang, FANG Chenxi, LIU Yating, WANG Yutong, XUE Linlei, SHI Jiaxin, REN Tianfang, ZHANG Junwei, BAO Manzhu, ZHANG Jie. Construction of Transient Transformation System of Petals in Prunus mume[J]. Acta Horticulturae Sinica, 2025, 52(4): 908-920.

图/表 13

图1 ‘雪梅’梅花4个发育阶段花朵形态气球期：花蕾松动、但花瓣未展开；初开期：部分花瓣稍微展开；碗状期：花瓣半展开；盛开期：花瓣完全展开且充盈丰富

Fig. 1 Flower morphology at four developmental stages of Prunus mume‘Xuemei’ Balloon stage：The buds are loose but the petals are not spread；Initial blooming stage：Some of the petals are slightly spread；Bowl stage：The petals are half spread；Blooming stage：The petals are fully spread and abundant

表1 本研究中所用引物序列

Table 1 Primers sequence used in this study

引物名称Primer	序列（5′-3′）Sequence
ANS-T	F：ATGGTGAGCTCTGATTCAGTGG；R：TCACTTGTTGAGCAGAGCCTC
pBI-PmANS	F：GAGAACACGGGGGAC TCTAGAATGGTGAGCTCTGATTCAGTGGA；
pBI-PmANS	R：ATAAGGGACTGACCA CCCGGGTCACTTGTTGAGCAGAGCCTCTT
ANS（121）	F：ATGGTGAGCTCTGATTCAGTGG；R：ACTTTTCCCGGCAATAACATACGG
ANS-1-2	F：CCAGGCCTCTGGCAAAATTC；R：GGCCAAATGGACAAGTCACG

表2 梅花‘雪梅’花瓣不同侵染方式下GUS瞬时表达率、材料坏死率和GUS等级占比

Table 2 GUS transient expression rate，material necrosis rate and GUS grade proportion of Prunus mume‘Xuemei’ petals under different infection modes

侵染方式 Infection mode	GUS瞬时表达率/% GUS transient expression rate	材料坏死率/% Material-necrosis rate	GUS-1等级占比/% GUS-1 grade proportion	GUS-2等级占比/% GUS-2 grade proportion	GUS-3等级占比/% GUS-3 grade proportion
活体注射In vivo injection	49.33 ± 9.23 ab	18.67 ± 6.11 ab	37.33 ± 10.01 a	10.67 ± 4.61 ab	1.33 ± 1.01 a
带枝水培Hydroponics with branches	49.34 ± 10.58 a	12.00 ± 4 ab	38.67 ± 8.33 a	4.00 ± 3.93 b	6.67 ± 4.62 a
整花真空Whole flower vacuum	66.67 ± 18.33 a	21.33 ± 6.11 a	33.33 ± 6.11 a	24.00 ± 13.86 a	9.33 ± 8.33 a
花瓣真空Petal vacuum	10.67 ± 9.24 b	0 b	10.67 ± 9.24 b	0 b	0 a

图2 不同侵染方式处理下梅花花瓣GUS染色效果

Fig. 2 Effects of different infection modes on GUS transient expression in Prunus mume petals

图3 不同侵染时间下梅花花瓣GUS染色效果

Fig. 3 Effects of different infection time on GUS transient expression in Prunus mume petals

表3 梅花‘雪梅’花瓣不同侵染时间下GUS瞬时表达率、材料坏死率和GUS等级占比

Table 3 GUS transient expression rate，material necrosis rate and GUS grade proportion of‘Xuemei’petals under different infection time

侵染时间/min Infection time	GUS瞬时表达率/% GUS transient expression rate	材料坏死率/% Material necrosis rate	GUS-1等级占比/% GUS-1 grade proportion	GUS-2等级占比/% GUS-2 grade proportion
10	34.67 ± 2.31 a	20.00 ± 6.11 a	34.67 ± 2.31 a	0 b
15	45.34 ± 6.66 a	13.33 ± 4.00 a	38.67 ± 8.33 a	6.67 ± 4.62 a
20	42.67 ± 9.24 a	29.33 ± 6.11 a	42.67 ± 9.24 a	0 b
30	45.33 ± 10.07 a	29.33 ± 6.11 a	45.33 ± 10.07 b	0 b

表4 梅花‘雪梅’花瓣不同发育阶段下GUS瞬时表达率、材料坏死率和GUS等级占比

Table 4 GUS transient expression rate，material necrosis rate and GUS grade proportion of Prunus mume‘Xuemei’petals under different development stages

发育阶段 Development stage	GUS瞬时表达率/% GUS transient expression rate	材料坏死率/% Material necrosis rate	GUS-1等级占比/% GUS-1 grade proportion	GUS-2等级占比/% GUS-2 grade proportion	GUS-3等级占/% GUS-3 grade proportion
气球期Balloon stage	49.33 ± 8 b	17.33 ± 2.31 a	45.33 ± 2.31 a	4.00 ± 0 a	0 a
初开期Initial blooming stage	77.33 ± 10.07 a	20.00 ± 2.31 ab	57.33 ± 10.07 a	14.67 ± 12.22 a	5.33 ± 5.11 a
碗状期Bowl stage	66.67 ± 2.31 b	17.33 ± 2.31 c	57.33 ± 9.24 a	5.33 ± 5.11 a	4.00 ± 0 a
盛开期Blooming stage	49.33 ± 9.24 b	6.67 ± 4.62 bc	46.67 ± 6.11 a	2.67 ± 2.31 a	0 a

图4 不同发育阶段处理下梅花花瓣GUS染色效果

Fig. 4 Effects ofdifferent development stages on GUS transient expression in Prunus mume petals

图5 梅花23个基因型花瓣的GUS瞬时表达率和材料坏死率 A ~ W：‘曹王黄香’‘淡粉垂枝’‘白阁宫粉’‘变瓣大红’‘二绿萼’‘小骨里红’‘六瓣红’‘粉台垂枝’‘江南台阁’‘小红朱砂’‘磨山小梅’‘三轮玉蝶’‘淡寒红’‘江南’‘粉妆台阁’‘寒红’‘变绿萼’‘无锡单杏’‘六瓣梅’‘台阁宫粉’‘铁骨红’ ‘多萼朱砂’‘乌羽玉’，下同。同组数据内不同小写字母表示在0.05水平差异显著

Fig. 5 GUS transient expression rate and material necrosis rate in Prunus mume petals of different genotypes A-W：‘Caowang Huangxiang’，‘Danfen Chuizhi’，‘Baige Gongfen’，‘Bianban Dahong’，‘Erlü’e’，‘Xiaoguli Hong’，‘Liuban Hong’，‘Fentai Chuizhi’，‘Jiangnan Taige’，‘Xiaohong Zhusha’，‘Moshan Xiaomei’，‘Sanlun Yudie’，‘Danhanhong’，‘Jiangnan’，‘Fenzhuang Taige’，‘Hanhong’，‘Bianlü’e’，‘Wuxi Danxing’，‘Liubanmei’，‘Taige Gongfen’，‘Tieguhong’，‘Duo’e Zhusha’，‘Wuyuyu’，the same below. Different lowercase letters in the same data indicate a significant difference at the 0.05 level

图6 不同基因型对梅花花瓣GUS染色效果

Fig. 6 Effects of different genotypes on GUS transient expression in Prunus mume petals

表5 不同侵染条件对‘雪梅’花瓣GUS瞬时表达率和材料坏死率的影响

Table 5 Effects of different infection conditions on GUS transient expression rate and material necrosis rate of‘Xuemei’petals

编号 No.	菌株类型 Agrobacterium strain	OD₆₀₀	共培养时间/d Co-cultivation time	GUS瞬时表达率/% GUS transient expression rate	材料坏死率/% Material necrosis rate
1	EHA105	0.4	1	0 c	0 d
2	EHA105	0.6	5	32.67 ± 5.03 a	28.00 ± 2.00 b
3	EHA105	0.8	3	28.33 ± 11.15 ab	25.00 ± 3.61 bc
4	GV3101	0.4	5	20.67 ± 2.31 b	26.67 ± 5.13 bc
5	GV3101	0.6	3	26.00 ± 3.00 ab	21.00 ± 2.00 c
6	GV3101	0.8	1	0 c	0 d
7	EHA105	0.4	3	27.00 ± 7.21 ab	21.33 ± 4.93 bc
8	EHA105	0.6	1	0 c	0 d
9	EHA105	0.8	5	26.00 ± 2.65 ab	37.33 ± 7.02 a
K1(G)	20.333	15.890	0
K2(G)	15.557	19.557	27.110
K3(G)	17.667	18.110	26.447
R(G)	4.776	3.667	27.110
K1(M)	17.667	16.000	0
K2(M)	15.890	16.333	22.443
K3(M)	19.553	20.777	30.667
R(M)	3.663	4.777	30.667

图7 9个正交处理下梅花花瓣GUS染色效果 1~9：对应正交试验编号处理下GUS染色结果

Fig. 7 Effects of different orthogonal treatments on GUS transient expression in Prunus mume petals 1-9：GUS staining results under the corresponding orthogonal experiment numbered treatments

图8 梅花花瓣瞬时转化后的表型观测（A）及半定量RT-PCR检测结果（B）

Fig. 8 Phenotype observation after transient transformation of Prunus mume petals（A）and semi-quantitative RT-PCR detection results（B）

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