CN113308385B - A Corynebacterium javanica strain DMC01 and its application in the control of East Asian locusts - Google Patents

本发明属于微生物学技术领域，具体涉及的是一种爪哇棒束孢菌株DMC01及其在防治东亚飞蝗上的应用。一种爪哇棒束孢菌株DMC01，其分类学名为Isaria javanica，于2021年4月28日保藏于广东省微生物菌种保藏中心，保藏号为GDMCC No.61632，其基因序列如SEQ ID No.3所示。本发明的爪哇棒束孢DMC01对东亚飞蝗具有较高致病力，可有效杀死东亚飞蝗，且具有较高僵虫率。本发明的爪哇棒束孢DMC01是一株在东亚飞蝗防治上具有潜在意义的生物防治菌株。

The invention belongs to the technical field of microbiology, and specifically relates to a Corynebacterium javanica strain DMC01 and its application in preventing and controlling the East Asian migratory locust. A Corynebacterium javanica strain DMC01, whose taxonomic name is Isaria javanica, was deposited in the Guangdong Provincial Microbial Culture Collection Center on April 28, 2021, and the deposit number is GDMCC No.61632, and its gene sequence is such as SEQ ID No. 3 shown. The Corynesporum javanica DMC01 of the present invention has high pathogenicity to the East Asian locust, can effectively kill the East Asian migratory locust, and has a high rate of mussels. The Corynebacteria javanica DMC01 of the present invention is a biological control strain with potential significance in the control of East Asian locusts.

一种爪哇棒束孢菌株DMC01及其在防治东亚飞蝗上的应用A Corynebacterium javanica strain DMC01 and its application in the control of East Asian locusts

技术领域technical field

本发明属于微生物学技术领域，具体涉及的是一种爪哇棒束孢菌株DMC01及其在防治东亚飞蝗上的应用。The invention belongs to the technical field of microbiology, and specifically relates to a Corynebacterium javanica strain DMC01 and its application in preventing and controlling the East Asian locust.

背景技术Background technique

东亚飞蝗Locusta migratoria manilensis(Meyen)属昆虫纲，直翅目，蝗科，别名蚂蚱、蝗虫。在中国，东亚飞蝗广泛分布于华北、华中、华东、西南、华南及台湾等，而黄淮海地区为常发区。东亚飞蝗为迁飞性、杂食性大害虫，主要取食禾本科和莎草科等杂草和作物，其中作物以取食玉米、水稻、甘蔗等，给各地的农业生产带来巨大的危害。东亚飞蝗危害的形成离不开其繁殖速度快、生殖后代数量多、迁飞性强、食性广、食量大等生理学和生态学特性。目前，国内外防治蝗虫灾害的措施包括化学防治、生态防治和生物防治手段，其中以化学防治为主要的防治手段。化学防治虽然有高效、速效、使用方便、经济效益高等优点，但其污染环境、杀伤天敌、对人畜有害以及容易引起害虫的再猖獗等缺点都不容忽视。East Asian locust Locusta migratoria manilensis (Meyen) belongs to Insecta, Orthoptera, Acrididae, alias grasshopper, locust. In China, East Asian migratory locusts are widely distributed in North China, Central China, East China, Southwest China, South China and Taiwan, etc., while the Huanghuaihai area is a frequent occurrence area. The East Asian migratory locust is a migratory and omnivorous large pest. It mainly feeds on weeds and crops such as Gramineae and Cyperaceae. Among them, crops feed on corn, rice, sugarcane, etc., which bring huge harm to agricultural production in various places. . The formation of East Asian migratory locust damage is inseparable from its physiological and ecological characteristics, such as fast reproduction speed, large number of reproductive offspring, strong migratory ability, wide food habits, and large food intake. At present, domestic and foreign measures to prevent and control locust disasters include chemical control, ecological control and biological control, among which chemical control is the main control method. Although chemical control has the advantages of high efficiency, quick effect, convenient use and high economic benefits, its shortcomings such as polluting the environment, killing natural enemies, being harmful to humans and animals, and easily causing the rampant pests cannot be ignored.

蝗虫有许多天敌，如捕食性昆虫芫菁、蜂虻等和捕食性鸟类百灵、云雀和粉红椋鸟等，但目前利用蝗虫天敌防治蝗灾的成熟技术和高效技术并不多。现有技术已有报道利用球孢白僵菌(Beauveria bassiana)和绿僵菌(Metarhizium)等虫生真菌防治东亚飞蝗。目前，还尚未见有利用爪哇棒束孢(Isaria javanica)防治东亚飞蝗的相关报道。Locusts have many natural enemies, such as predatory insects such as coriander, bee flies, etc., and predatory birds such as lark, lark, and pink starling. However, there are not many mature and efficient technologies for using locust natural enemies to control locust disasters. In the prior art, it has been reported that insect-borne fungi such as Beauveria bassiana and Metarhizium are used to control East Asian migratory locusts. At present, there is no relevant report on the use of Isaria javanica to control the East Asian locust.

公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

发明内容SUMMARY OF THE INVENTION

本发明的目的是提供一种爪哇棒束孢菌株DMC01及其在防治东亚飞蝗上的应用。The purpose of the present invention is to provide a kind of Corynebacterium javanica strain DMC01 and its application on the control of East Asian locust.

一种爪哇棒束孢菌株DMC01，其分类学名为Isaria javanica，其基因序列如SEQID No.3所示。A Corynebacterium javanica strain DMC01, its taxonomic name is Isaria javanica, and its gene sequence is shown in SEQID No.3.

本发明提供的种爪哇棒束孢菌株DMC01是通过采集天然感病的昆虫，分离纯化得来，经形态学和分子生物学鉴定为爪哇棒束孢Isaria javanica，于2021年4月28日保藏于保藏于广东省微生物菌种保藏中心，(简称GDMCC，地址：广州市先烈中路100号大院59号楼5楼，广东省微生物研究所，邮编：510070)，保藏号为GDMCC No.61632。The Corynebacterium javanica strain DMC01 provided by the present invention is obtained by collecting natural susceptible insects, separating and purifying, and is identified as Corynebacterium javanica Isaria javanica by morphology and molecular biology, and is preserved on April 28, 2021 in It is preserved in the Guangdong Provincial Microbial Culture Collection Center, (GDMCC for short, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Institute of Microbiology, zip code: 510070), and the preservation number is GDMCC No.61632.

本发明还提供了所述爪哇棒束孢菌株DMC01在防治东亚飞蝗上的应用，所属技术领域的技术人员，可以将爪哇棒束孢菌株DMC01制成微生物杀虫剂，用于防治东亚飞蝗。The invention also provides the application of the Corynebacterium javanica strain DMC01 in preventing and controlling the East Asian migratory locust. Those skilled in the art can make the Corynebacterium javanica strain DMC01 into a microbial insecticide for preventing and controlling the East Asian migratory locust. .

与现有技术相比，本发明具有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:

(1)本发明对菌株进行分离纯化，通过形态学观察和分子鉴定，确定获得的菌株DCM01为爪哇棒束孢Isaria javanica；其次申请人还测定了其对东亚飞蝗的毒力，经测定发现本发明的爪哇棒束孢DMC01对东亚飞蝗具有较高致病力，可有效杀死东亚飞蝗，且具有高僵虫率。(1) The present invention separates and purifies the bacterial strain, and through morphological observation and molecular identification, it is determined that the obtained bacterial strain DCM01 is Isaria javanica; Secondly, the applicant has also measured its virulence to the East Asian locust, and found through the measurement The Corynebacteria javanica DMC01 of the invention has high pathogenicity to the East Asian locust, can effectively kill the East Asian migratory locust, and has a high rate of mussels.

(2)本发明的爪哇棒束孢DMC01是一株在东亚飞蝗的防治上具有潜在意义的生物防治菌株，可开发成微生物杀虫剂，为东亚飞蝗的绿色防控提供技术支撑。(2) Corynebacteria javanica DMC01 of the present invention is a biological control strain with potential significance in the prevention and treatment of East Asian locusts, and can be developed into a microbial insecticide to provide technical support for the green prevention and control of East Asian migratory locusts.

保藏信息说明Description of preservation information

Isaria javanica DMC01，保藏编号为GDMCC No.61632，保藏日期为2021年4月28日，保藏单位为广东省微生物菌种保藏中心(GDMCC)，保藏地址为广州市先烈中路100号大院59号楼5楼。Isaria javanica DMC01, the preservation number is GDMCC No.61632, the preservation date is April 28, 2021, the preservation unit is the Guangdong Provincial Microbial Culture Collection Center (GDMCC), and the preservation address is Building 59, Yard, No. 100, Xianlie Middle Road, Guangzhou 5th Floor.

附图说明Description of drawings

图1为DMC01菌株菌落形态正面图；Fig. 1 is the front view of the colony morphology of DMC01 strain;

图2为DMC01菌株菌落形态背面图；Fig. 2 is the back view of DMC01 bacterial strain colony morphology;

图3为DMC01菌株的系统发育图；Fig. 3 is the phylogenetic diagram of DMC01 strain;

图4为DMC01菌株在东亚飞蝗上的侵染状；Fig. 4 is the infection state of DMC01 strain on East Asian migratory locusts;

具体实施方式Detailed ways

下面结合具体的实施例对本发明做进一步的详细说明，所述是对本发明的解释而不是限定。The present invention will be further described in detail below in conjunction with specific embodiments, which are to explain rather than limit the present invention.

下述实施例中所用的材料和试剂，如无特殊说明，均可从商业途径得到。下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified. The experimental methods used in the following examples are conventional methods unless otherwise specified.

实施例Example

一、爪哇棒束孢菌株DMC01的分离纯化1. Isolation and purification of Corynebacterium javanica strain DMC01

采集广西壮族自治区钦州市钦廉林场的桉大毛虫僵虫虫体，在超净台上，将僵虫浸入75％的酒精中表面消毒30s后用0.1％升汞浸泡消毒3min，然后依次转移到3瓶无菌水中冲洗，取出置于灭过菌的滤纸上把虫体解剖成小块；The worms of Eucalyptus caterpillars were collected from Qinlian Forest Farm, Qinzhou City, Guangxi Zhuang Autonomous Region. On the ultra-clean bench, the worms were immersed in 75% alcohol for surface disinfection for 30s, then soaked and disinfected with 0.1% mercuric chloride for 3 minutes, and then transferred to the Rinse with 3 bottles of sterile water, take it out and place it on sterilized filter paper to dissect the worm into small pieces;

将组织块放置在加入50ug/mL的链霉素的PDA培养基上，每皿放5块，然后置于25℃下培养，待其长出菌丝后挑取转出菌株放到PDA平板上进行纯化培养，纯化培养重复3次。将筛选得到的一株真菌编号为DMC01，并挑取纯化得到的菌丝接入PDA斜面试管中，待菌株长满斜面试管后置于4℃的环境下作为菌种保存。The tissue pieces were placed on the PDA medium with 50ug/mL streptomycin, 5 pieces per dish, and then cultured at 25°C. After the hyphae were grown, the strains were picked out and placed on the PDA plate. Purification culture was performed, and the purification culture was repeated 3 times. One strain of fungus obtained by screening was numbered as DMC01, and the hyphae obtained by picking and purification were inserted into the PDA slanted tube.

二、爪哇棒束孢菌株DMC01鉴定2. Identification of Corynebacterium javanica strain DMC01

25℃下培养的菌株，在培养过程中对菌落进行观察，菌落的形态如图1和图2所示。For the strains cultivated at 25°C, the colonies were observed during the cultivation process, and the morphology of the colonies was shown in Figures 1 and 2.

从图1和图2可以看出，在PDA平板上，菌落为圆形，菌丝白色，质地为厚绒毛状，菌落背面淡黄色，孢子层颜色为浅紫灰色。As can be seen from Figure 1 and Figure 2, on the PDA plate, the colony is round, the hyphae are white, the texture is thick villi, the back of the colony is light yellow, and the color of the spore layer is light purple-gray.

在显微镜下，菌丝无色透明，分子孢子梗上着生2-4个瓶梗形成的轮生体，瓶梗基部椭圆形膨大，向上逐渐变细；分生孢子在瓶梗上生长形成孢子链；分生孢子呈透明光滑的长椭圆形。Under the microscope, the hyphae are colorless and transparent, and the sporophore has 2-4 whorls of phialides. The base of the phial is elliptical and swollen, and gradually tapers upward; the conidia grow on the phial to form a spore chain. ; Conidia are transparent and smooth oblong.

根据上述菌落形态和孢子形态等特征，初步鉴定菌株为爪哇棒束孢Isariajavanica。According to the above characteristics of colony morphology and spore morphology, the strain was preliminarily identified as Isariajavanica.

三、分子鉴定3. Molecular identification

1.按照康为世纪NuClean Plant Genomic DNA Kit试剂盒的说明书提取DNA，得到爪哇棒束孢菌株DMC01的基因组DNA。1. Extract DNA according to the instructions of Kangwei Century NuClean Plant Genomic DNA Kit to obtain the genomic DNA of Corynebacterium javanica strain DMC01.

2.采用真菌TUB引物Bt2a和Bt2b对目的基因β-微管蛋白(β-tubulin，TUB2)进行扩增，得到PCR扩增产物；其中，引物Bt2a和Bt2b的序列见下表1，PCR反应体系见下表2。2. Use fungal TUB primers Bt2a and Bt2b to amplify the target gene β-tubulin (β-tubulin, TUB2) to obtain PCR amplification products; wherein, the sequences of primers Bt2a and Bt2b are shown in Table 1 below, and the PCR reaction system See Table 2 below.

表1引物Bt2a和Bt2b的序列Table 1 Sequences of primers Bt2a and Bt2b

表2 PCR反应体系Table 2 PCR reaction system

PCR反应程序：95℃变性3min；95℃变性30s、52℃退火30s、72℃延伸1min，共35个循环；72℃延伸10min。PCR reaction program: denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s, annealing at 52 °C for 30 s, extension at 72 °C for 1 min, a total of 35 cycles; extension at 72 °C for 10 min.

3.将扩增产物回收纯化、连接转化并进行DNA测序，得到其核苷酸序列见SEQ IDNo.3。3. Recover and purify the amplified product, ligate and transform it, and perform DNA sequencing to obtain its nucleotide sequence as shown in SEQ ID No. 3.

4.将核苷酸序列与NCBI中已知序列进行BLAST对比，构建DMC01菌株系统发育树图，结果见图3。4. The nucleotide sequence was compared with the known sequence in NCBI by BLAST to construct a phylogenetic tree diagram of the DMC01 strain. The results are shown in Figure 3.

从图3可以看出，DMC01菌株与AY624224(Isaria javanica)聚为一类，并且其形态特征符合爪哇棒束孢Isaria javanica的特征，确定菌株DMC01为爪哇棒束孢Isariajavanica。因此，将其命名为Isaria javanica DMC01。It can be seen from Figure 3 that the DMC01 strain is clustered with AY624224 (Isaria javanica), and its morphological characteristics conform to the characteristics of Isaria javanica, confirming that the strain DMC01 is Isaria javanica. Hence, it is named Isaria javanica DMC01.

四、爪哇棒束孢菌株DMC01对东亚飞蝗的毒力测定4. Toxicity determination of Corynebacterium javanica strain DMC01 against East Asian locusts

1.用接种环将分离纯化得到的爪哇棒束孢菌株分生孢子刮入50mL的三角瓶里，加入20mL含有0.05％的吐温-80水溶液，充分摇匀后用血球计数板在显微镜下计数，得到初始浓度孢子母液，将孢子液配制成1×10⁵、1×10⁶、1×10⁷、1×10⁸个/mL共4个浓度梯度，每个浓度为一个处理，以0.05％的吐温-80水溶液为对照，每个处理设3组重复，每个重复15头东亚飞蝗。1. Scrape the conidia of Corynebacterium javanica strain obtained by separation and purification into a 50 mL conical flask with an inoculating loop, add 20 mL of 0.05% Tween-80 aqueous solution, shake well and count under a microscope with a hemocytometer , obtain the initial concentration of spore mother liquid, and prepare the spore liquid into 4 concentration gradients of 1×10 ⁵ , 1×10 ⁶ , 1×10 ⁷ , 1×10 ⁸ /mL in total, each concentration is a treatment, with 0.05% The Tween-80 aqueous solution was used as the control, and each treatment was repeated in 3 groups, with 15 East Asian locusts for each repetition.

2.将配好的各浓度孢子液以及对照处理的0.05％的吐温-80水溶液均匀喷洒在东亚飞蝗身上，以喷至孢子液不滴下为止，然后单头将东亚飞蝗放入养虫皿中，置于25℃培养箱内光暗各12h进行培养，每天用新鲜的玉米叶片喂养，并每天定时观察、记录各处理的具体死亡情况，连续观察8d，发现死虫则将其移除培养皿，放入放有滤纸并灭过菌的玻璃培养皿中保湿培养，观察其是否长出菌丝，结果见图4。2. Evenly spray the prepared spore solution of each concentration and the 0.05% Tween-80 aqueous solution of the control treatment on the East Asian locust until the spore solution does not drip, and then put the East Asian locust into the insect raising with a single head. In a dish, placed in a 25 °C incubator for 12 h in light and dark, and fed with fresh corn leaves every day, and observed and recorded the specific death situation of each treatment at regular intervals every day, and observed continuously for 8 days. If dead insects were found, they were removed. The petri dish was placed in a glass petri dish with filter paper and sterilized for moisturizing and cultivation, and it was observed whether the mycelium grew. The results are shown in Figure 4.

从图4可以看出，DMC01菌株侵染东亚飞蝗后，虫体上会长出菌丝。It can be seen from Figure 4 that after the DMC01 strain infects the East Asian locust, hyphae will grow on the worm.

记录僵虫的头数，并计算不同浓度爪哇棒束孢DMC01对四龄东亚飞蝗的校正死亡率以及不同浓度爪哇棒束孢DMC01处理后的四龄东亚飞蝗僵虫率，结果见下表3和4。Record the number of zombies, and calculate the corrected mortality rate of the fourth-instar E. migratory locust with different concentrations of DMC01 and the rate of fourth-instar E. migratory locusts treated with different concentrations of DMC01. The results are shown in the table below. 3 and 4.

表3不同浓度爪哇棒束孢DMC01对四龄东亚飞蝗的校正死亡率Table 3 Corrected mortality of the fourth-instar E. migratory locust with different concentrations of Corynebacterium javanica DMC01

从表3可以看出，孢子浓度越高，对东亚飞蝗的校正死亡率越高；在最低浓度1×10⁵个/mL下接种8d后，其校正死亡率也达到了69.05％；随着接种时间的延长，东亚飞蝗的校正死亡率升高，同一孢子浓度处理下，接种8d的校正死亡率要高于接种4d的校正死亡率。It can be seen from Table 3 that the higher the spore concentration, the higher the corrected mortality rate of East Asian locusts; after 8 days of inoculation at the lowest concentration of 1 × 10 ⁵ /mL, the corrected mortality rate also reached 69.05%; With the prolongation of inoculation time, the corrected mortality rate of East Asian locust increased. Under the same spore concentration, the corrected mortality rate of inoculation 8 days was higher than that of inoculation 4 days.

表4不同浓度爪哇棒束孢DMC01处理后四龄东亚飞蝗僵虫率Table 4 The rate of the fourth instar E. migratory locust after treatment with different concentrations of Corynebacterium javanica DMC01

从表4可以看出，爪哇棒束孢DMC01处理后的东亚飞蝗的僵虫率高，最低浓度1×10⁵个/mL处理下的僵虫率为44.44％，而1×10⁷个/mL和1×10⁸个/mL处理下的僵虫率均为100％。From Table 4, it can be seen that the rate of larvae in the East Asian migratory locust treated with DMC01 was high, and the rate of larvae under the lowest concentration of 1×10 ⁵ /mL was 44.44%, while the rate of 1×10 ⁷ /mL was 44.44%. The rates of zombies under the treatments of mL and 1×10 ⁸ /mL were both 100%.

结合表3和表4可以看出，本发明的爪哇棒束孢DMC01对东亚飞蝗有高致病力，1×10⁷个/mL以上浓度的孢子液就可以有效杀死东亚飞蝗，且具有高僵虫率。As can be seen in conjunction with Table 3 and Table 4, Conychosporium javanica DMC01 of the present invention has high pathogenicity to East Asian migratory locusts, and the spore liquid of more than 1 × 10 ⁷ /mL concentration can effectively kill East Asian migratory locusts, and Has a high rate of zombies.

综上所述，本发明的爪哇棒束孢DMC01对东亚飞蝗具有较高致病力，可有效杀死东亚飞蝗，且具有高僵虫率，是一株在东亚飞蝗的防治上具有潜在意义的生物防治菌株。To sum up, DMC01 of Corynebacteria javanica of the present invention has high pathogenicity to East Asian migratory locust, can effectively kill East Asian migratory locust, and has a high mussels rate, and is a strain with high virulence in the control of East Asian migratory locust. Potentially significant biological control strains.

前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式，并且很显然，根据上述教导，可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用，从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. These descriptions are not intended to limit the invention to the precise form disclosed, and obviously many changes and modifications are possible in light of the above teachings. The exemplary embodiments were chosen and described for the purpose of explaining certain principles of the invention and their practical applications, to thereby enable others skilled in the art to make and utilize various exemplary embodiments and various different aspects of the invention. Choose and change. The scope of the invention is intended to be defined by the claims and their equivalents.

序列表 sequence listing

<110> 广西大学<110> Guangxi University

<120> 一种爪哇棒束孢菌株DMC01及其在防治东亚飞蝗上的应用<120> A Corynebacterium javanica strain DMC01 and its application in the control of East Asian migratory locusts

<130> 2021<130> 2021

<160> 3<160> 3

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 24<211> 24

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 1<400> 1

ggtaaccaaa tcggtgctgc tttc 24ggtaaccaaa tcggtgctgc tttc 24

<210> 2<210> 2

<211> 24<211> 24

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 2<400> 2

accctcagtg tagtgaccct tggc 24accctcagtg tagtgaccct tggc 24

<210> 3<210> 3

<211> 352<211> 352

<212> DNA<212> DNA

<213> 爪哇棒束孢(Isaria javanica)<213> Isaria javanica

<400> 3<400> 3

gtaaccaaat cggtgctgct ttctggcaga ccatctctgg cgagcacggc ctcgactcca 60gtaaccaaat cggtgctgct ttctggcaga ccatctctgg cgagcacggc ctcgactcca 60

gcggtgtcta caatggcact tccgagcttc agctcgagcg catgaatgtc tacttcaacg 120gcggtgtcta caatggcact tccgagcttc agctcgagcg catgaatgtc tacttcaacg 120

aggtttgcta taccggcaca acgcgtagct tggcttcatt gtggatacta accgcgaatt 180aggtttgcta taccggcaca acgcgtagct tggcttcatt gtggatacta accgcgaatt 180

tcttcaggcc tctggtaaca agtatgttcc tcgcgccgtc ctcgtcgatc ttgagcccgg 240tcttcaggcc tctggtaaca agtatgttcc tcgcgccgtc ctcgtcgatc ttgagcccgg 240

taccatggac gctgtccgtg ccggtccctt cggtcagctc ttccgccccg acaacttcgt 300taccatggac gctgtccgtg ccggtccctt cggtcagctc ttccgccccg acaacttcgt 300

tttcggtcag tccggtgctg gcaacaactg ggccaagggt cactacactg ag 352tttcggtcag tccggtgctg gcaacaactg ggccaagggt cactacactg ag 352