CN110412263B - 一种快速高效的茶树质体型谷氨酰胺合成酶基因定位方法 - Google Patents

一种快速高效的茶树质体型谷氨酰胺合成酶基因定位方法 Download PDF

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CN110412263B
CN110412263B CN201910700388.9A CN201910700388A CN110412263B CN 110412263 B CN110412263 B CN 110412263B CN 201910700388 A CN201910700388 A CN 201910700388A CN 110412263 B CN110412263 B CN 110412263B
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刘美雅
张群峰
倪康
石元值
马立峰
伊晓云
汤丹丹
阮建云
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Abstract

本发明提出了一种快速高效的茶树质体型谷氨酰胺合成酶基因定位方法，试验当天将新鲜幼嫩的茶树叶片样品置于PBS缓冲液中，琼脂糖凝胶包埋固定后，于震荡切片机中切成50‑100μm切片，获得的茶树叶片横切片分别与茶树CsGS2特异抗体、商业抗体二抗杂交，最后于激光共聚焦显微镜下拍照。本发明细胞学基因定位方法能够快速高效的进行基因的精细定位；基因的定位可达到细胞学水平；试验过程所需周期短；避免因在模式植物拟南芥上进行茶树的基因定位带来的异源困扰；安全、可操作性和重复性强。

Description

一种快速高效的茶树质体型谷氨酰胺合成酶基因定位方法
技术领域
本发明涉及基因定位技术领域，具体涉及一种快速高效的茶树质体型谷氨酰胺合成酶基因定位方法。
背景技术
基因的组织定位很大程度上决定了其所行使的功能。如茶树氮素代谢限制酶编码基因谷氨酰胺合成酶基因，有定位于细胞质中的胞质型谷氨酰胺合成酶GS1和在叶绿体或线粒体中的质体型谷氨酰胺合成酶GS2。前者主要在根部氮代谢中发挥作用，而后者则主要将叶绿体和光呼吸再合成的NH4 +转化成谷氨酰胺，同时也参与根部氮的合成。拟南芥5个GS1成员由于表达部位的不同而导致其对不同氮源的响应不同，如GLN1;1、 GLN1;2 、GLN1;3、 GLN1;4根据他们是在根表皮细胞还是维管组织中表达而导致其对NH4 +响应存在极大的差异。
因此，对茶树基因进行精细定位是非常必要的。
茶树属多年生乔灌木，次生代谢物含量丰富，特别是多酚类含量物质高，可占总干物质重的20%-35%。正是由于其丰富的酚类物质含量导致其在遗传转化上的瓶颈，迄今并没有形成一个成熟的茶树遗传转化体系，导致茶树基因功能研究往往只能依靠在遗传转化体系成熟的拟南芥、番茄、烟草等植物中进行，如基因的定位、功能回复试验。但是异源转化并不能很好的解释茶树基因的确切功能，茶树的基因在别的物种中所起的调控作用是否与在茶树树体内一样并无充分的证据说明。
茶树为喜铵植物，研究已表明谷氨酰胺合成酶在其偏性吸收中起着重要调控作用，然而并不清楚是哪个编码基因在起着主导作用。另外，目前所报道的均为GS1基因，并无GS2相关基因的研究报道。对GS2基因进行精确定位，可进一步阐述茶树的氮吸收利用特性。
发明内容
为了解决上述的技术问题，本发明的目的是提供一种茶树质体型谷氨酰胺合成酶基因GS2的精细定位，以填补现有研究中对茶树GS2相关报道的空白，同时克服了现有技术中由于前处理时间长、操作要求高、且实验周期长的问题。
本发明提供一种快速高效的茶树质体型谷氨酰胺合成酶基因定位方法，试验当天将新鲜幼嫩的茶树叶片样品置于PBS缓冲液中，琼脂糖凝胶包埋固定后，于震荡切片机中切成50-100 μm切片，获得的茶树叶片横切片分别与茶树CsGS2特异抗体、商业抗体二抗杂交，最后于激光共聚焦显微镜下拍照。
作为本发明进一步的改进，所述PBS缓冲液的浓度为0.1 mol/L。
作为本发明进一步的改进，所述琼脂糖凝胶为5%的琼脂糖凝胶。
作为本发明进一步的改进，具体包括以下步骤：
S1. 采用RACE法获得CsGS2基因序列全长，如基因序列SEQ ID NO. ：1所示，氨基酸序列如SEQ ID NO. ：2所示，根据所获得的序列进行基因特异抗体的合成；
S2. 将茶树扦插苗，在完全营养液中进行水培，取新生长叶片于PBS缓冲液中；
S3. 配置琼脂糖凝胶，待凝胶温度到50-60℃时，将上述茶树组织置于凝胶中，随后室温冷却至凝胶凝固，此步骤是完成新鲜茶树组织的固定；
S4. 将固定好的茶树组织，置于振荡切片机上进行横切，切片厚度50-100 μm，切好的叶的横切片室温孵育于PBS缓冲液中；
S5. 用含5% BSA的磷酸缓冲液封闭30 分钟后，用PBS缓冲液冲洗四次；
S6. 在含基因特异性抗体的PBS缓冲液中杂交孵育，用PBS缓冲液冲洗四次；之后用含商业抗体Alexa Fluor 555 goat anti-rabbit IgG的PBS缓冲液中杂交孵育；最后再用PBS缓冲液多次冲洗；
S7. 将切片平铺于载玻片上，压片后于激光共聚焦显微镜下进行荧光观察并拍照。
作为本发明进一步的改进，步骤S3中所述琼脂糖凝胶为5%的琼脂糖凝胶。
作为本发明进一步的改进，步骤S4中所述PBS缓冲液的pH为7.4。
作为本发明进一步的改进，步骤S5和步骤S6中所述PBS缓冲液的浓度为0.1mol/L。
作为本发明进一步的改进，步骤S6中每次杂交孵育时间为1-2小时。
作为本发明进一步的改进，步骤S6中所述基因特异性抗体为茶树质体型谷氨酰胺合成酶基因CsGS2。
作为本发明进一步的改进，步骤S7中所述激光共聚焦显微镜为蔡司710双光子激光共聚焦显微镜。
本发明具有如下有益效果：
本发明细胞学基因定位方法适用于非模式植物茶树，遗传转化体系不完善，无法在茶树上进行基因的组织定位，同时也适用于其他遗传转化体系不完善的植物；
本发明细胞学基因定位方法能够快速高效的进行基因的精细定位；基因的定位可达到细胞学水平；试验过程所需周期短；避免因在模式植物拟南芥上进行茶树的基因定位带来的异源困扰；安全、可操作性和重复性强。
本发明与传统方法和实施效果的比较见表1：
表1 本发明与现有技术的主要优点
比较项目 本发明的技术特征 其他基因定位方法（原位杂交） 其他基因定位方法（转基因）
样品要求 茶树新鲜活体叶片 茶树组织经甲醛、丙酮脱水后的石蜡固定样 拟南芥、烟草
同源性 同源基因 同源基因 异源基因
技术要求 人为影响因素较小 操作人员熟练程度对实验处理效果影响较大 人为影响因素大
实验周期时间 效率高，一个白天可以完成实验 实验周期长，从样品固定到探针杂交至少需一个星期 实验周期最长2-3个月以上，即时采用模式植物也需要保证植株纯合才能进行后续的研究
操作安全性 本发明中所使用的试剂磷酸缓冲液对人体无毒害，且对环境安全 组织样品固定采用采用甲醛、二甲苯等毒性大 转基因存在生物安全问题
附图说明
图1为实施例1和实施例2中水培“龙井43”扦插苗。
图2为实施例1茶树幼嫩叶片脂糖包埋固定切片。
图3为实施例1中CsGS2在叶片中的细胞学定位显微图。
具体实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整的描述，显然，所述的实施例只是本发明的部分具有代表性的实施例，而不是全部实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的其他所有实施例都属于本发明的保护范围。
实施例1
茶树质体型谷氨酰胺合成酶基因CsGS2的定位
（1）CsGS2基因cDNA全长的克隆
取0.1 g一年生龙井43(Camelliasinensis cv.‘Longjing43’)扦插苗叶片采用RNAprep pure多糖多酚植物总RNA提取试剂盒(Tiangen, 北京)进行总RNA的提取。NanoDrop 2000微量核酸蛋白测定仪测定所提取RNA质量及浓度，同时用1.2%琼脂糖凝胶电泳检测总RNA完整性。根据前期得到的基因片段设计引物GS2-F:ATGGCACAGATTTTGGCTCCTT/GS2-R: TTAGACATTCATTGCCAGTT。采用Clontech公司的SMARTer™ RACE cDNA Amplification Kit对该基因cDNA全长进行克隆。
（2）抗体合成由生物公司完成（杭州华安生物技术有限公司）
（3）‘龙井43’茶树一年生扦插苗完全展开叶，5% 琼脂糖凝胶包埋固定后，在振荡切片机（VT 1000 S，Leica，Bensheim，Germany）上进行横切，切片厚度50-100 μm，切好的根、茎、叶的横切片室温孵育于pH为7.4的PBS缓冲液中，CsGS2抗体由杭州华安生物技术有限公司制备，兔二抗IgG购自Sigma公司。经一抗和二抗杂交孵育后平铺于载玻片上，压片后于710双光子激光共聚焦显微镜下进行荧光观察并拍照（图3），其中箭头指示该基因定位于质体中。
具体步骤如下：
S1. 采用RACE法获得CsGS2基因序列全长，如基因序列SEQ ID NO. ：1所示，氨基酸序列如SEQ ID NO. ：2所示，根据所获得的序列进行基因特异抗体的合成；
S2. 将一年生龙井43(Camelliasinensis cv.‘Longjing43’)扦插苗（图1），在完全营养液中进行水培，取新生长叶片于0.1mol/LPBS缓冲液中；
S3. 配置5%琼脂糖凝胶，待凝胶温度到50-60℃时，将上述茶树组织置于凝胶中，随后室温冷却至凝胶凝固，此步骤是完成新鲜茶树组织的固定（图2）；
S4. 将固定好的茶树组织，置于振荡切片机上进行横切，切片厚度50-100 μm，切好的叶的横切片室温孵育于pH为7.4的PBS缓冲液中；
S5. 用含5% BSA的磷酸缓冲液封闭30 min后，用0.1mol/LPBS缓冲液冲洗四次；
S6. 在含茶树质体型谷氨酰胺合成酶基因CsGS2特异抗体的0.1mol/LPBS缓冲液中杂交孵育，时间为1-2小时，用0.1mol/LPBS缓冲液冲洗四次；之后用含商业抗体AlexaFluor 555 goat anti-rabbit IgG的0.1mol/LPBS缓冲液中杂交孵育，时间为1-2小时；最后再用0.1mol/LPBS缓冲液多次冲洗；
S7. 将切片平铺于载玻片上，压片后于蔡司710双光子激光共聚焦显微镜下进行荧光观察并拍照（图3）。
与现有技术相比，本发明细胞学基因定位方法适用于非模式植物茶树，遗传转化体系不完善，无法在茶树上进行基因的组织定位，同时也适用于其他遗传转化体系不完善的植物；
本发明细胞学基因定位方法能够快速高效的进行基因的精细定位；基因的定位可达到细胞学水平；试验过程所需周期短；避免因在模式植物拟南芥上进行茶树的基因定位带来的异源困扰；安全、可操作性和重复性强。
本领域的技术人员在不脱离权利要求书确定的本发明的精神和范围的条件下，还可以对以上内容进行各种各样的修改。因此本发明的范围并不仅限于以上的说明，而是由权利要求书的范围来确定的。
序列表
<110> 中国农业科学院茶叶研究所
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<213> 茶树CsGS2基因序列(人工序列)
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Ala Thr Gly Thr Ala Ala Cys Ala Cys Cys Ala Thr Ala Cys Ala Cys
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Ala Ala Gly Ala Cys Ala Ala Thr Cys Thr Cys Gly Ala Ala Gly Cys
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Thr Thr Ala Cys Thr Cys Cys Ala Ala Ala Cys Ala Ala Ala Cys Gly
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Thr Ala Thr Cys Ala Gly Ala Cys Gly Cys Thr Cys Ala Thr Thr Ala
755 760 765
Thr Ala Ala Gly Gly Cys Thr Thr Gly Cys Thr Thr Gly Thr Ala Thr
770 775 780
Gly Cys Cys Gly Gly Ala Ala Thr Thr Ala Ala Cys Ala Thr Thr Ala
785 790 795 800
Gly Thr Gly Gly Cys Ala Cys Cys Ala Ala Cys Gly Gly Ala Gly Ala
805 810 815
Ala Gly Thr Cys Ala Thr Gly Cys Cys Thr Gly Gly Cys Cys Ala Gly
820 825 830
Thr Gly Gly Gly Ala Ala Thr Thr Thr Cys Ala Ala Gly Thr Ala Gly
835 840 845
Gly Thr Cys Cys Ala Ala Gly Thr Gly Thr Gly Gly Gly Ala Ala Thr
850 855 860
Thr Gly Ala Ala Gly Cys Thr Gly Gly Ala Gly Ala Thr Cys Ala Cys
865 870 875 880
Ala Thr Gly Thr Gly Gly Thr Gly Cys Gly Cys Thr Ala Gly Ala Thr
885 890 895
Ala Cys Ala Thr Cys Cys Thr Thr Gly Ala Gly Ala Gly Ala Ala Thr
900 905 910
Thr Ala Cys Thr Gly Ala Ala Cys Ala Ala Gly Cys Thr Gly Gly Thr
915 920 925
Gly Thr Thr Gly Thr Thr Cys Thr Cys Thr Cys Ala Cys Thr Cys Gly
930 935 940
Ala Thr Cys Cys Gly Ala Ala Ala Cys Cys Ala Ala Thr Ala Gly Ala
945 950 955 960
Gly Gly Gly Thr Gly Ala Cys Thr Gly Gly Ala Ala Cys Gly Gly Thr
965 970 975
Gly Cys Ala Gly Gly Ala Thr Gly Cys Cys Ala Cys Ala Cys Cys Ala
980 985 990
Ala Thr Thr Ala Cys Ala Gly Thr Ala Cys Ala Ala Ala Gly Ala Gly
995 1000 1005
Thr Ala Thr Gly Ala Gly Ala Gly Ala Gly Gly Ala Thr Gly Gly Cys
1010 1015 1020
Gly Gly Gly Thr Thr Thr Gly Ala Ala Gly Thr Ala Ala Thr Thr Ala
1025 1030 1035 1040
Ala Gly Ala Ala Ala Gly Cys Ala Ala Thr Thr Cys Thr Gly Ala Ala
1045 1050 1055
Thr Cys Thr Thr Thr Cys Gly Cys Thr Thr Cys Gly Thr Cys Ala Cys
1060 1065 1070
Ala Ala Ala Gly Ala Ala Cys Ala Cys Ala Thr Cys Ala Gly Thr Gly
1075 1080 1085
Cys Thr Thr Ala Thr Gly Gly Ala Gly Ala Ala Gly Gly Ala Ala Ala
1090 1095 1100
Thr Gly Ala Ala Ala Gly Ala Ala Gly Gly Thr Thr Gly Ala Cys Gly
1105 1110 1115 1120
Gly Gly Ala Ala Ala Gly Cys Ala Thr Gly Ala Ala Ala Cys Thr Gly
1125 1130 1135
Cys Cys Ala Gly Cys Ala Thr Cys Ala Ala Cys Ala Cys Ala Thr Thr
1140 1145 1150
Cys Thr Cys Thr Thr Gly Gly Gly Gly Ala Gly Thr Cys Gly Cys Thr
1155 1160 1165
Ala Ala Thr Cys Gly Cys Gly Gly Thr Thr Gly Thr Thr Cys Ala Ala
1170 1175 1180
Thr Cys Cys Gly Cys Gly Thr Gly Gly Gly Gly Cys Gly Thr Gly Ala
1185 1190 1195 1200
Ala Ala Cys Thr Gly Ala Gly Ala Ala Gly Cys Ala Ala Gly Gly Cys
1205 1210 1215
Ala Ala Ala Gly Gly Thr Thr Ala Cys Thr Thr Gly Gly Ala Ala Gly
1220 1225 1230
Ala Thr Ala Gly Gly Cys Gly Thr Cys Cys Gly Gly Cys Thr Thr Cys
1235 1240 1245
Ala Ala Ala Cys Ala Thr Gly Gly Ala Cys Cys Cys Ala Thr Ala Thr
1250 1255 1260
Ala Thr Thr Gly Thr Gly Ala Cys Gly Gly Cys Cys Thr Thr Ala Thr
1265 1270 1275 1280
Thr Gly Gly Cys Thr Gly Ala Ala Ala Cys Thr Ala Cys Cys Thr Thr
1285 1290 1295
Ala Cys Thr Gly Thr Gly Gly Gly Ala Gly Cys Cys Thr Ala Cys Gly
1300 1305 1310
Cys Thr Cys Gly Ala Gly Gly Cys Thr Gly Ala Gly Cys Thr Cys Thr
1315 1320 1325
Thr Gly Cys Thr Gly Cys Thr Cys Ala Gly Ala Ala Ala Cys Thr Gly
1330 1335 1340
Gly Cys Ala Ala Thr Gly Ala Ala Thr Gly Thr Cys Glu Ala Ala Ala
1345 1350 1355 1360
Ala Thr Cys Ala Gly Thr Cys Gly Gly Ala Ala Ala Ala Ala Gly Ala
1365 1370 1375
Ala Gly Cys Thr Cys Thr Cys Ala Thr Ile Cys Cys Thr Ala Thr Thr
1380 1385 1390
Thr Thr Ala Thr Thr Thr Cys Thr Cys Ala Gly Ala Thr Cys Thr Thr
1395 1400 1405
Thr Ala Gly Gly Gly Ala Ala Gly Ala Thr Cys Ala Cys Thr Thr Gly
1410 1415 1420
Thr Ala Ala Ala Thr Ala Ala Gly Thr Cys Cys Cys Cys Ala Gly Thr
1425 1430 1435 1440
Gly Gly Thr Thr Cys Thr Thr Ala Gly Ala Ala Gly Cys Ala Thr Ala
1445 1450 1455
Thr Thr Ala Gly Ala Cys Cys Thr Thr Gly Gly Gly Ala Thr Cys Ala
1460 1465 1470
Gly Gly Gly Thr Gly Thr Gly Thr Thr Cys Ala Thr Gly Ala Ala Thr
1475 1480 1485
Ala Thr Thr Thr Gly Cys Cys Ala Gly Ala Thr Gly Ala Ala Gly Gly
1490 1495 1500
Ala Cys Ala Thr Thr Gly Cys Ala Cys Thr Gly Thr Thr Gly Thr Ala
1505 1510 1515 1520
Ala Gly Cys Ala Ala Thr Thr Gly Ala Thr Thr Ala Thr Thr Ala Ala
1525 1530 1535
Thr Thr Thr Thr Ala Thr Thr Ile Thr Thr Ala Gly Ala Thr Ala Ala
1540 1545 1550
Gly Gly Ala Ala Thr Cys Cys Cys Cys Cys Ala Ala Ala Cys Gly Gly
1555 1560 1565
Gly Thr Thr Cys Thr Cys Thr Gly Gly Ala Cys Cys Ala Ala Ala Ala
1570 1575 1580
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
1585 1590 1595 1600
Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala
1605 1610
<210> 2
<211> 432
<212> PRT
<213> 茶树CsGS2氨基酸序列(人工序列)
<400> 2
Met Ala Gln Ile Leu Ala Pro Ser Pro Gln Trp Gln Met Arg Val Thr
1 5 10 15
Lys Asn Ser Thr Asn Ala Ser Pro Met Thr Ser Thr Met Trp Ser Ser
20 25 30
Leu Phe Leu Lys Gln Asn Lys Lys Ala Thr Ala Lys Thr Phe Ala Lys
35 40 45
Phe Arg Val Phe Ala Leu Gln Ser Glu Asn Ser Thr Ile Asn Arg Met
50 55 60
Glu Asp Leu Leu Ser Met Asp Val Thr Pro Tyr Thr Asp Lys Ile Ile
65 70 75 80
Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Ile Asp Leu Arg Ser Lys
85 90 95
Ser Lys Thr Ile Ser Lys Pro Val Lys His Pro Ser Glu Leu Pro Lys
100 105 110
Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro Gly Glu Asp Ser
115 120 125
Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp Pro Phe Arg Gly
130 135 140
Gly Asn Asn Ile Leu Val Ile Cys Asp Thr Tyr Thr Pro Gln Gly Glu
145 150 155 160
Pro Ile Pro Thr Asn Lys Arg Tyr Lys Ala Ala Glu Ile Phe Ser Asn
165 170 175
Lys Lys Val Val Asp Glu Ile Pro Trp Tyr Gly Ile Glu Gln Glu Tyr
180 185 190
Thr Leu Leu Gln Thr Asn Val Lys Trp Pro Leu Gly Trp Pro Val Gly
195 200 205
Gly Tyr Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ala Ala Gly Ala Asp
210 215 220
Lys Ser Phe Gly Arg Asp Ile Ser Asp Ala His Tyr Lys Ala Cys Leu
225 230 235 240
Tyr Ala Gly Ile Asn Ile Ser Gly Thr Asn Gly Glu Val Met Pro Gly
245 250 255
Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile Glu Ala Gly Asp
260 265 270
His Met Trp Cys Ala Arg Tyr Ile Leu Glu Arg Ile Thr Glu Gln Ala
275 280 285
Gly Val Val Leu Ser Leu Asp Pro Lys Pro Ile Glu Gly Asp Trp Asn
290 295 300
Gly Ala Gly Cys His Thr Asn Tyr Ser Thr Lys Ser Met Arg Glu Asp
305 310 315 320
Gly Gly Phe Glu Val Ile Lys Lys Ala Ile Leu Asn Leu Ser Leu Arg
325 330 335
His Lys Glu His Ile Ser Ala Tyr Gly Glu Gly Asn Glu Arg Arg Leu
340 345 350
Thr Gly Lys His Glu Thr Ala Ser Ile Asn Thr Phe Ser Trp Gly Val
355 360 365
Ala Asn Arg Gly Cys Ser Ile Arg Val Gly Arg Glu Thr Glu Lys Gln
370 375 380
Gly Lys Gly Tyr Leu Glu Asp Arg Arg Pro Ala Ser Asn Met Asp Pro
385 390 395 400
Tyr Thr Val Thr Ala Leu Leu Ala Glu Thr Ile Leu Leu Trp Glu Pro
405 410 415
Thr Leu Glu Ala Glu Ala Leu Ala Ala Gln Lys Leu Ala Met Asn Val
420 425 430

Claims (1)

1.一种快速高效的茶树质体型谷氨酰胺合成酶基因定位方法，其特征在于，试验当天将新鲜幼嫩的茶树叶片样品置于PBS缓冲液中，琼脂糖凝胶包埋固定后，于震荡切片机中切成50-100μm切片，获得的茶树叶片横切片分别与茶树CsGS2特异抗体、商业抗体二抗杂交，最后于激光共聚焦显微镜下拍照；
所述PBS缓冲液的浓度为0.1mol/L；
所述琼脂糖凝胶为5％的琼脂糖凝胶；
包括以下步骤：
S1.采用RACE法获得CsGS2基因序列全长，如氨基酸序列如SEQ ID NO.：2所示，根据所获得的序列进行基因特异抗体的合成；
S2.将茶树扦插苗，在完全营养液中进行水培，取新生长叶片于PBS缓冲液中；
S3.配置琼脂糖凝胶，待凝胶温度到50-60℃时，将上述茶树组织置于凝胶中，随后室温冷却至凝胶凝固，此步骤是完成新鲜茶树组织的固定；
S4.将固定好的茶树组织，置于振荡切片机上进行横切，切片厚度50-100μm，切好的叶的横切片室温孵育于PBS缓冲液中；
S5.用含5％BSA的磷酸缓冲液封闭30分钟后，用PBS缓冲液冲洗四次；
S6.在含基因特异性抗体的PBS缓冲液中杂交孵育，用PBS缓冲液冲洗四次；之后用含商业抗体Alexa Fluor 555goat anti-rabbit IgG的PBS缓冲液中杂交孵育；最后再用PBS缓冲液多次冲洗；
S7.将切片平铺于载玻片上，压片后于激光共聚焦显微镜下进行荧光观察并拍照；
步骤S3中所述琼脂糖凝胶为5％的琼脂糖凝胶；
步骤S4中所述PBS缓冲液的pH为7.4；
步骤S5和步骤S6中所述PBS缓冲液的浓度为0.1mol/L；
步骤S6中每次杂交孵育时间为1-2小时；
步骤S6中所述基因特异性抗体为茶树质体型谷氨酰胺合成酶基因CsGS2；
步骤S7中所述激光共聚焦显微镜为蔡司710双光子激光共聚焦显微镜。
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