Читать книгу Biotechnology of Fruit and Nut Crops - Группа авторов - Страница 174

In the last decade, molecular and genomic approaches have been applied in pineapple to study genes involved in fruit ripening, nematode infection, the CAM pathway and other biological processes (Moyle et al., 2005a,b; Ong et al., 2012; Zhang et al., 2014). Moyle et al. (2005a) obtained more than 5600 ESTs and developed an online bioinformatics resource, PineappleDB (Moyle et al., 2005b; Fig. 5.1.1). Relative EST clone abundance in green and yellow fruit libraries correlated well with mRNA abundance in their respective tissues as shown by northern analysis. A number of genes upregulated during fruit ripening were identified; among the most interesting were two metallothionein genes and a MAD-box gene. Following this work, Moyle and Botella (2014) analysed 4102 EST sequences from the vascular cylinder tissue of pineapple roots at early and late stages of infection with the root-knot nematode Meloidogyne javanica. Northern analysis and quantitative real-time PCR confirmed a metallothionein-like protein, an alpha tubulin, a phosphoglycerate mutase, a glyceraldehyde phosphate dehydrogenase and a mannose-binding lectin differentially expressed during gall formation.

Fig. 5.1.1. PineappleDB, the online pineapple bioinformatics resource (www.pgel.com.au). View of a typical entry containing database information.

Ong et al. (2012) sequenced the transcriptome of extracts from ripe yellow flesh using the Illumina next-generation sequencing platform with the assembly producing 28,728 unique transcripts with a mean length of c.200 bp. Koia et al. (2012) developed a 9277-element pineapple microarray and used it to profile the gene expression changes that occur during the ripening of pineapple fruit. Microarray analyses identified 271 unique cDNAs that were differentially expressed, by at least 1.5 times, between the mature green and mature yellow stages of ripening.

Botella and Smith (2008) reported an earlier EST project devised to study gene expression in roots after root-knot nematode infestation; 4102 EST sequences were obtained, including 1298 early infection clones, 2461 late infection clones and 343 non-infected root tip clones. Northern analysis and quantitative real-time PCR identified a variety of genes differentially expressed during gall formation.

Nashima et al. (2015) sequenced the complete pineapple chloroplast genome using the 454 GS FLX+ and Illumina platform. The total length was 159,636 bp, which comprised a pair of inverted repeats (each 26,774 bp) separated by a small single-copy region of 18,622 bp and a large single-copy region of 18,466 bp. Their analysis predicted 113 unique genes (79 protein-coding, 4 rRNA and 30 tRNA genes) and 19 duplicated genes in the inverted repeats. Almost identical data were reported by Redwan et al. (2015) who sequenced the chloroplast genome combining the PacBio long sequence reads, which have a relatively high error rate, with short, but very accurate reads from the Illumina system. This group predicted a slightly higher number of coding regions of 117.

The sequencing of the pineapple genome by Ming et al. (2015) has triggered a number of publications based on bioinformatics analyses including the genome-wide identification and analysis of proteolytic enzyme genes, nucleotide-binding site (NBS) resistance genes, microRNAs (miRNAs), carbon flux and carbohydrate gene families, miRNAs and phased small interfering RNAs (phasiRNAs) (Paull et al., 2016; Wai et al., 2016a; Zhang et al., 2016). These studies provide important genetic and biochemical information. For example, 512 pineapple genes encoding putative proteolytic enzymes have been identified providing an important resource for the discovery of new proteases for medical use (Wai et al., 2016a). NBS resistance genes are important for providing immunity against pathogens and 177 of these genes were identified that were distributed across 20 pineapple linkage groups. The analysis of genes involved in carbohydrate biosynthesis, breakdown and modification revealed the presence of several glycosyltransferases, glycoside hydrolases, carbohydrate esterases and polysaccharide lyases (Paull et al., 2016). Wai et al. (2016b) used a combined approach by mapping the transcript data generated with next-generation sequencing technology and available ESTs and mRNAs in the public database to the newly sequenced pineapple genome to identify 5146 genes which generated alternatively spliced isoforms. The annotated information of these data provides a resource for further characterization of these genes and their biological roles and can be accessed at the Plant Alternative Splicing Database (http://proteomics.ysu.edu/altsplice/). The analysis of miRNAs and phasiRNAs combining computational and experimental approaches discovered 131 conserved miRNAs grouped into 37 families and 16 novel miRNAs. Computer analysis was employed to predict the putative targets of the miRNAS and phasiRNAs (Zheng et al., 2016). Yusuf et al. (2015) used high-throughput small RNA sequencing in a Solexa platform to identify miRNAs by homology with existing entries in the miRBase database revealing the presence of 153 miRNAs from 41 miRNA families.

The draft genome of ‘MD-2’ pineapple, the most popular cultivar for fresh consumption, has been independently obtained by Redwan et al. (2015), who used a combination of the PacBio long sequencing reads and the accurate Illumina short reads to achieve 99.6% of genome coverage. They also used RNAseq to identify differentially expressed genes in ripening pineapple fruits, discovering a large proportion of enriched ethylene-related transcripts and hypothesizing that this hormone has a role in the ripening of the (non-climacteric) pineapple fruit. This hypothesis had been previously proposed by Cazzonelli et al. (1998) who cloned the ACC synthase and ACC oxidase genes, involved in ethylene synthesis, and proved that they were highly induced in fruits during the ripening process.

The study of genes involved in natural (premature) flowering has extraordinary importance given its commercial implications. Next-generation sequencing has been used to study the transcriptome of pineapple plants exposed to the flowering-inducing agent ethephon, which releases ethylene upon application to the plant (Liu and Fan, 2016). Several differentially expressed genes treated with ethephon were tentatively associated with floral induction, including homologues of ethylene response factors, the ETR1 ethylene receptor and genes associated with flowering in Arabidopsis, i.e. AP1, FT, VRN1 and AG. The expression profile of four ethylene receptors, two ETR2 homologues and two ERS1 homologues, in response to ethylene and ethephon application, has been determined using quantitative real-time PCR with the ETR2 homologues being the most sensitive to ethylene treatment (Li et al., 2016).

An important prerequisite for pineapple biotechnology is the availability of adequate promoters but, unfortunately, few pineapple promoters have been characterized. To identify constitutively expressed pineapple promoters, the translation factor SUI1 and the ribosomal protein L36 promoters were cloned and studied in transgenic Arabidopsis plants where they were able to drive constitutive transgene expression (Koia et al., 2013). The promoter of the AcMADS1 transcription factor, which is strongly induced during pineapple fruit ripening was cloned and proven to direct strong β-glucuronidase (GUS) expression in fruits and developing floral organs in tomato and Arabidopsis, suggesting that AcMADS1 may play a role in flower development as well as in fruitlet ripening (Moyle et al., 2014). Phylogenetic analysis positioned AcMADS1 in the same superclade as LeMADS-RIN, a master regulator of fruit ripening upstream of ethylene in climacteric tomato. LeMADS-RIN has been proposed to be a global ripening regulator shared among climacteric and non-climacteric species, but AcMADS1 failed to complement the tomato rin mutation in transgenic tomato plants (Moyle et al., 2014).