The coding region of CnFatB1, CnFatB2, and CnFatB3 of coconut was cloned in E.coli DH5α by heat shock method using pMD18-T. After identifying validated positive clones, FatB gene family was sequenced. The nucleotide sequence of CnFatB1 was related to C.nucifera acyl-ACP thioesterase FatB1 with a nodal value of 99, CnFatB2 showed that was related to C. nucifera acylACP thioesterase FatB2 with a nodal value of 97 and CnFat3 was related to C. nucifera acyl-ACP thioesterase FatB3 with 98 for a nodal value respectively. Expressed vector was constructed by ligating CnFatB1, CnFatB2 and CnFatB3 to linearized pCAMBIA 1302 and transformed into E.coli by heat shock. Moreover, expression vector was also transformed into Agrobacterium tumefaciens GV3103. Potential colonies harboring FatB genes were screened by colony PCR, and confirmed by using expression vector as template for PCR and by enzyme digestion using BglII and BstEII. All results showed that CnFatB1, CnFatB2 and CnFatB3 were in expected size.

Latt ZK, Yang Y, Li J (2017) Cloning and Sequencing Analyses of FATB Gene Family from Coconut (Cocos nucifera). Int J Plant Biol Res 5(4): 1073.

•    Coconut
•    Acyl-ACP thioesterase
•    Fat B
•    E.coli DH5α
•    Agrobacterium tumefaciens

Coconut is one of the world’s most versatile economically important tropical crops and is well known for its commercial and industrial applications in tropical and subtropical areas, such as in food and beverages and as a source of wood and handicrafts [1].

Oil extracted from coconut pulp is widely applied in cooking, soaps and cosmetics. Coconut is one of the few plants that store medium chain-length fatty acids (MCFAs) as the major portion of their energy reserves in the endosperm of seeds. In developed coconut fruit, more than 83.92% of the oil consists of MCFAs and long-chain fatty acids (C12:0, C14:0, C16:0 and C18:0), the majority of which is lauric acid (C12:0) ranging from 47.48% to 50.5% [2]. Coconut also has more MCFAs than soybean [3], oil palm and safflower, and animal fats such as butter, tallow, fish oil and lard [4-8]. It was also reported that MCFAs are abundant in the oil produced in fruits of coconut (i.e. predominantly C12 and C14 and a small amount (0.2-1%) of C6 fatty acids [9-11]. Thioesterases play a pivotal role in fatty acid synthesis owing to their role in catalysing the terminal reaction of fatty acid biosynthesis, which regulates the fatty acid composition of storage lipids, especially in plant seeds [12,13]. The expression of thioesterase genes displayed the highest levels in expanding tissues that are typically very active in lipid biosynthesis, such as developing seed endosperm and young expanding leaves [14,15]. The enzyme that determines fatty acid chain length is acyl-acyl carrier protein thioesterase (acyl-ACP TE). This enzyme catalyzes the terminal reaction of fatty acid biosynthesis, acyl-ACP thioester bond hydrolysis to release a free fatty acid and ACP. In discrete phyla and/or tissues of specific organisms (primarily higher plant seeds), thioester hydrolysis optimally produces medium-chain (C8-C14) fatty acids (MCFAs), which have wide industrial applications (e.g., producing detergents, lubricants, cosmetics, and pharmaceuticals) [16]. TEs that specifically hydrolyze medium- chain acyl-ACP substrates have been studied widely [16-18]. Several plant acyl-ACP TEs (e.g. CnFatB3) produced significant amounts of unsaturated fatty acids (UFAs) when expressed in E. coli. These include 10:1, 12:1, 14:1, and 16:1 fatty acids, which do not usually accumulate in E. coli or in the original host plant tissues from which the acyl-ACP TE was isolated [19].

Plant acyl-ACP thioesterases fall into two distinct but related classes known as FatA and FatB [20,21]. Thioesterases, known as oleoyl-ACP thioesterases, that prefer oleoyl-ACP as a substrate with limited activity on 16:0- and 18:0-ACP define the FatA class. These thioesterases are assumed to be ubiquitousin plant tissues and responsible for the fatty acid composition found in membranes and most temperate plant oils. The FatB class contains all of the specialized thioesterases described to date from plants which accumulate specific saturated fatty acids, as well as an apparently ubiquitous thioesterase with a broader specificity for longer chain saturated and unsaturated substrates with a preference for 16:0-ACP and an unknown in vivo function. It is notable that a distinct acyl-ACP in vivo functions. It is notable that a distinct acyl-ACP thioesterase has been cloned with a preference for each substrate from 8:0-ACP through 18:1-ACP with the exception of 18:0-ACP, although an 18:0-ACP thioesterase has been biochemically characterized from leek epidermal tissue [22]. Enzymes from both the FatA and FatB classes have limited activity on 18:0-ACP, but none of them prefer that substrate. Three acyl-ACP thioesterases; CnFatB1 (JF338903), CnFatB2 (JF338904), CnFatB3 (JF338905)) from coconut have been isolated and characterised [13].

The objectives of this research work were to clone FatB family gene of Coconut; CnFatB1, CnFatB2 and CnFatB3 and analyze the sequencing of these three genes.

Isolation of FatB gene family

To isolate FatB gene family of coconut, the coding region of CnFatB1, CnFatB2 and CnFatB3 were isolated by PCR using pMD18-T which contains these gene families as a template with respective primers as shown in (Table 1). The primers used for isolation and cloning of CnFatB1, CnFatB2 and CnFatB3 were designed by using Vector NTI. The conditions of PCR were as follows; cycling conditions of 30 rounds of amplification; initial denaturation at 94ºC for 5min,denaturation at 94ºC for 30 sec, annealing at 56ºC for 30 sec and extension at 72ºC for 1.20 min followed by final extension at 94ºC for 7min. The PCR products were checked on a 1% agarose gel.

Transformation of pMD18-T-FatB into competent E.coli DH5α

The PCR products of FatB genes were checked by agarose gel electrophoresis and recovered from the gel by DNA purification kit. The amplified products of CnFatB1, CnFatB2 and CnFatB3 were ligated to pMD18-T. 5µl of solution I, 4 µl of DNA and 1 µl of pMD18-T were put into tube, mixed and incubated the mixture at 4ºC overnight. It was designated as pMD18-T-FatB. It was transformed into competent E.coli DH5α by heat shock method.

50µl of competent E.coli was mixed with 5 µl of ligation mixture and put on ice for 30 min. After 30 min, the mixture was incubated at 42ºC for 90 seconds, immediately transferred into ice and let for 1-2 min. 500 µl of Luria-Bertani (LB) broth was added to the mixture and incubated at 37ºC for 45min by shaking at 180 rpm. After that, the culture broth was centrifuged at 8000 rmp for 1 min and the supernatant was discarded after taking 100 µl of supernatant. The pellet was resuspended in 100 µl of LB and spread on LB media containing ampicillin. Finally the plates were incubated at 37ºC overnight. After overnight incubation, it was observed for the appearance of colonies.

Valiadation of FatB gene family by bacterial clone analysis and enzyme digestion

Potential bacterial clones harboring FatB genes were screened using colony PCR. Single E. coli colonies were picked up using a sterile toothpick and added to a 20µl PCR reaction mixture with respective primers for CnFatB1, CnFatB2, and CnFatB3. The bacteria were identified by PCR.

pMD18-T-FatB was extracted from E.coli DH5α after inoculation of single colony of E.coli DH5α into LB broth containing ampicillin at 37ºC for overnight. The DNA extracted from identified positive colonies was validated with double enzyme digestion using BstEII and BglII.

Sequencing and phylogenetic analysis

Sequencing of CnFatB1, CnFatB2 and CnFatB3 was performed by selecting validated positive colonies of E.coli. The phylogenetic tree was constructed by computing the lowest BIC value. The parameter of K2 +G was 59 in the use of a neighbor-joining method in MEGA6. Bootstrap with 500 replicates was used to establish the confidence limit of the tree branches.

Construction of expression vector

Plant expression vector pCAMBIA1302 was digested with BglII and BstEII and two bands, GFP gene (~538bp) and the remaining part of vector were checked by 1% agarose gel electrophoresis. GFP gene was deleted from the vector after enzyme digestion.

pMD18-T-FatB was also digested with the same enzymes. After enzyme digestion, FatB genes (1200 bp) were purified by DNA purification kit.

For enzyme digestion, 1µl of 10xNE buffer, 0.2µg of DNA and 0.2 µl of BglII were mixed and filled H2 O to final 10µl. The reaction mixture was incubated at 37ºC for 15 min. After 15 min incubation, 0.2µl of BstEII was added and incubated again at 60ºC for 15 min. After enzyme digestion, DNA in expected size of pCAMBIA1302 and CnFatB1, CnFatB2, and CnFatB3 were visualized by agarose gel electrophoresis and purified by DNA purification kit.

The purifiedCnFat1, CnFatB2 and CnFatB3 were ligated to linearized pCAMBIA1302 under the reaction of T4 DNA ligase. The reaction mixture used was shown in (Table 2).

The reaction mixture was incubated at 16ºC for overnight and transformed into competent E.coli DH5α as described in above procedure. The recombinant expression vector was validated by colony PCR using the same conditions as described above.

Transformation of A. tumefaciens with expression vector

Expression vector was transformed into A.tumefaciens by freeze thaw method. A single colony of the Agrobacterium strain was inoculated into 3 ml of LB in a 15 ml snap-cap tube. Growth was allowed at 30°C for overnight by shaking at 200 rpm. For A.tumefaciens GV3101, gentamycin or rifampicin was added to LB media. Overnight culture was inoculated into 50 ml of LB in a 250 ml flask with 0.5 ml (1/100 volume) and incubated at 30°C until mid-log (OD600 was between 0.5 and 1.0). This took ~4-5 hours to get the cells to this stage. The culture was chilled 5-10 minutes on ice, centrifuged at 3000 rpm for 5 minutes at 4°C in chilled sterile centrifuge tubes. Supernatant was discarded; the tubes were inverted for 30-60 seconds. Pellet was resuspended in 1 ml of ice cold 20 mM CaCl2 . 0.1 ml of bacterial suspension was dispended into each of two pre-chilled 1.5 ml. microfuge tubes on ice. One is a control.

1 ug of plasmid DNA was added to one tube and nothing to the other the control and mixed well. The tubes were breezed in liquid N2 , thenthe tubes were allowed to thaw for 5 minutes at 37°C.

1 ml of LB was added to each tube, and incubated for ~2 hours by shaking at 200 rpm of 30°C. Culture broth was centrifuge at 8000 rpm for 1 min, supernatant was removed. Pellets were resuspended in 100 µl of LB and spread onto LB containing Kanamycin and Gentamycin. All of the suspension were plated on appropriate antibiotic-LB plates and incubate for two days at 30°C. Transformed colonies should be visible on the second day of incubation.

Table 1: Primers used for isolation and validation after cloning of CnFatB1, CnFatB2, CnFatB3 of coconut (Coco snucifera).

Table 2: Reaction conditions for ligation of CnFatB1, CnFatB2, CnFatB3 into linearized 1302 under T4 DNA ligase.

Isolation of FatB gene

The coding region of CnFatB1, CnFatB2 and CnFatB3 of coconut were amplified using pMD18-T as template with respective primers and 1200 bp in size of each gene were obtained as shown in (Figure 1).

Cloning of FatB genes

Figure 2 showed the general procedure for cloning of CnFatB genes of coconut using pMD18-T and pCAMBIA after constructing as expression vector by cloning in E.coli DH5α. Purified CnFatB1, CnFatB2 and CnFatB3 were ligated to pMD18-T and transformed into E.coli DH5α. Potential colonies harboring FatB genes were screened by colony PCR. Two positive colonies for CnFatB1, four positive colonies for CnFatB2 and two colonies for CnFatB3 were obtained. It was shown in (Figure 3a).

pMD18-T-Fat B was extracted from E.coli and used as template to validate the FatB genes in plasmid by using the same primers. CnFatB1, CnFatB2 and CnFatB3 were in uniform size from colony PCR and from using pMD18-T-FatB as template. It was shown in (Figure 3b).

After enzyme digestion of pD18-T-FatB with BglII and BstEII, the products were visualized by 1% agarose gel electrophoresis. CnFatB1, CnFatB2 and CnFatB3 were seen in expected size as shown in (Figure 4). The results showed that the size of CnFatB1, CnFatB2 and CnFatB3 were in consistent with the expected size and also consistent from all examination

Sequencing and phylogenetic tree analysis

By sequencing, CnFatB1 was related to Cocos nucifer FatB1 with a nodal value of 99, CnFatB2 was related to Cocos nucifer FatB2 with 97 for a nodal value and CnFatB3 was related to Cocos nucifer FatB3 with a nodal value of 98. Phylogenetic analysis was conducted according to the similarity of the FatA and FatB conserved domain sequences from different species. The analysis indicated that CnFatB1 was highly related to CnFatB1 (JX275886.1 and JF338903.1) of Cocos nucifera, CnFatB2 was highly related to CnFatB2 (JF338904.1) of Cocos nucifera, and CnFatB3 was highly related to CnFatB3 (JF338905.1) of Cocos nucifera. The phylogenetic tree was shown in (Figure 5).

Construction of expression vector and transformation in E.coli

After digestion of pMD18-T-FatB and pCAMBIA1302 with BglII and BstEII, CnFatB1, CnFatB2, CnFatB3 were ligated to linearized pCMABIA1302 in which GFP was deleted as shown in (Figure 6) and transformed into E.coli. In this study, it was not needed to use GFP as reporter gene. But GFP excited by ultraviolet light will emit green fluorescence light [23]. GFP gene employed as reportor gene is widely applied in plant genetic transformation [24-26] due to its advantages of stable fluorescent light emission, convenient detection and non-species specificity, no poisoning to plant as well as being capable of in vivo detection [27].

Growth of colonies on LB media containing Kanamycin was shown in (Figure 7). Potential colonies harboring FatB genes were screened by colony PCR and expression vector was digested with BglII and BstEII. CnFatB1, CnFatB2 and CnFatB3 were seen in expected size from both methods. It was shown in (Figure 8ab).

Jing et al., (2011) cloned Acyl-ACP TE cDNAs from endosperm of nearly mature coconuts, constructed expression vector pUC57 and transformed into E.coli strain K27. Each TE expressed in E.coli strain K27 and free fatty acids accumulated in the medium were extracted and analyzed in vitro. It was also reported that it appears that CnFatB1, CnFatB2, and CnFatB3 might work together to determine the fatty acid composition of coconut oil, which contains primarily 12:0 (43-50%) and 14:0 (16-22%) and small amounts of 6:0, 8:0, and 10:0 fatty acids [9-11].

CocoFatB1 was isolated from coconut endosperm during fruit development and transformed into Tobacco by A.tumefaciens. Fatty acid composition of transgenic tobacco seeds was increased [28].

Transformation of expression vector into A. tumefaciens GV3103

FatB genes are important gene family that can determine the chain length of fatty acid. Therefore, many researchers studied the role of FatB genes by transforming into other species using different transformation methods. In this study, it was also studied the cloning ofCnFatB gene family in A.tumefaciens and validated their cloning by PCR for the purpose of transformation to other species. Expression vector was transformed into A.tumefaciens Gv3103 and growth of A.tumefaciens on LB media containing Kanamycin and Gentamycin was shown in (Figure 9a). Potential colonies harboring FatB genes were checked by colony PCR. FatB1, FatB2 and FatB3 in expected size were seen in (Figure 9b).

CnFatB1, CnFatB2 and CnFatB3 were isolated and cloned in E.coli DH5α. After sequencing, isolated CnFatB1, CnFatB2 and CnFatB3 were highly related to CnFatB1 (JX275886.1 and JF338903.1) of Cocos nucifera, CnFatB2 was to CnFatB2 (JF338904.1) of Cocos nucifera, and CnFatB3 was highly related to CnFatB3 (JF338905.1) of Cocos nucifera). For construction of expression vector, pCAMBIA1302 was used after deletion of GFP. After transformation of expression vector in E.coli DH5α, some positive colonies were identified by colony PCR. In addition, it was confirmed that expression vector harboring FatB genes by using expression vector as template for PCR and enzyme digestion by BglII and BstEII. All results showed that CnFatB1, CnFatB2 and CnFatB3 were consistent in size with expected size.

This research work was supported by Ministry of Science and Technology, China as a Talented Young Scientist Award, the International Science and Technology Cooperation projects of Hainan Province (No. KJHZ2014-24) and the fundamental Scientific Research Funds for Chinese Academy of Tropical Agriculture Sciences (CATAS-No. 1630032012044, 1630052014002, and 1630152017019).

The author would like to thank Ministry of Science and Technology, China for financial support while staying in China, and Director, Coconut Research Institute, Chinese Academy of Sciences, China for allowing conducting this research work.

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