Transcriptomic Sequencing Analysis and Comparison of Biological Networks Involved in Grapevine Leaf and Fruit Development.pdf

1、The genera Vitis is one of the oldest commercially and economically important fruit crops cultivated over the globe, producing about 67.5 million tons of grape berries along with highly valued, value added and processed

2、products such as juices, jams, liquors and wines.Leaf and fruits are the major vegetative and reproductive organs for plant growth and multiplication of plants.Hence, understanding the ongoing biological activities in me

3、tabolic networks during these organs development is very important.Grape Berry and leaf development is a complex series of physical and biochemical changes, which are controlled by many metabolic networks.To some extent,

4、 the biophysical changes that occur through the multifaceted process of berry and leaf growth should be predisposed by the activities and presence of metabolic networks.These networks are essential to be controlled by th

5、e transcriptional regulation of mRNA.The understanding of these networks will not only provide the thoughtful knowledge of the essential processes that control grape berry growth and development, but also reveal insights

6、 into the genetic basis of grape berry quality that could potentially assist the wine industry.Recent advancements and development in high-throughput Sequencing Technology have become an essential technique in different

7、fields like digital gene expression profiling, epigenomics, genomics, and transcriptomics.These methodologies are used for dexterous sequencing of multiple RNA molecules and abandace of RNA molecules to be sequenced with

8、in a short period of time.In the present study, proceeding to illumina sequencing, determining RNA integrity numbers for poly RNA were separated from each of the four different developmental stages of cv.Summer Black lea

9、ves by using Illumina HiSeqTM 2000.The sum of 272,941,656 reads were generated from leaf at four different developmental stages, resulting more than 27 billion nucleotides of sequence data.RNA samples of each stage were

10、indexed through unique nucleic acid identifiers and sequenced.In the present study, we observed leaf development networks and the levels of different genes expression in comparison with the leaf initiation to maturity st

11、ages.In case of fruits,De-multiplexing using the unique identifiers discovered that data consisted of 28,808,716 (100％) clean reads from fruit sample of 40 DAF, 27,941,132 reads from fruit sample 65 DAF, and 25,457,565 r

12、eads from sample 90 DAF.From the total reads, total mapped reads (23.58％), total unmapped sequence reads (76.42％), unique matches (19.93％), multiple matches (3.65％) and perfect matches (13.73％) were recorded.KEGG annotat

13、ion of leaf depicted that the highest number of transcripts in 2Avs4A (2,963) followed by 1Avs4A (2,920), and 3Avs4A (2,294) out of 15,614 (71％) transcripts that were recorded.In comparison, sum of 1532 transcripts was a

14、nnotated in GOs,including Cellular component, with the highest number in “Cell part” 251 out of 353 transcripts (71.1％), followed by intracellular organelle 163 out of 353 transcripts (46.2％),while in molecular function

15、and metabolic process 375 out of 525 (71.4％)transcripts,multicellular organism process 40 out of 525 (7.6％) transcripts in biological process were the most common in 1Avs2A.In comparison for GOs in and among fruit growth

16、 at specific developmental stages, the highest transcripts were found in Biological process including cellular process of 2,350 and metabolic process of 2,251 followed by response to stimulus of 809 and biological regula

17、tion of 859 transcripts.In the comparison of Cellular component, we found the highest numver of transcripts in cell of 2,776 and organelle 1,962 followed by cell part of 2,776 transcripts, while in case of Molecular func

18、tion, we observed the highest number of transcripts in binding (2,329) followed by catalytic activity of 1,768 and localization 493 transcripts.In grapevine, during the initial stages of leaf development, 1Avs2A showed o

19、nly a single transcript related to photosystem Ⅰ (reaction center subunit PSI-N, chloroplast,putative/PSI-N, putative (PSAN)) was down-regulated and none of them showed upregulation during this stage; although in compari

20、son between 1A and 4A Photosystem Ⅱ showed 5 transcripts including 4 up-regulated (reaction center family protein PsbP, CemA-like proton extrusion protein-related PetA, root FNR 2 PetH and photosystem Ⅱ eaction center pr

21、otein C PsbC) and a single transcript was down-regulated (photosynthetic electron transfer C PetC).However in fruits, 32 transcripts in the early stages of fruit growth including alcohol dehydrogenase (8), hydroxymethylg

22、lutaryl-CoA synthase (3), pyruvate decarboxylase (5), lipoxygenase (LOX) (3), carotenoid cleavage dioxygenase (9), were predicted.The network related to photosynthesis in leaf, 24 transcripts were identified during diffe

23、rent leaf growth stages involved photosystem Ⅰ reaction center subunit (5),photosystem Ⅱ reaction center protein (12).In case of fruit developmental networks, we found 32 transcripts during the early stages of fruit grow

24、th including alcohol dehydrogenase (8), hydroxymethylglutaryl-CoA synthase (3), pyruvate decarboxylase (5),lipoxygenase (LOX) (3) and carotenoid cleavage dioxygenase (9), 13 transcripts related to myb-related proteins we

25、re found, throughout the grape berry development and their expression pattems from veraison onwards were the same.The number of transcripts was differentially expressed, although the up regulation trend was increased til

26、l maturity of the berries.Anthocyanidin 5, 3-O-glucosyltransferase 8 transcripts were found and the expression trends were different during developmental phase, however the expression of the genes was decreased towards m

27、aturity.In the present study 11 sugar phosphate protein transcripts were detected, of which 4 were up regulated in young berries and the rest were differentially expressed, although the overall expression level decreased

28、 towards the maturity of the berries.We found 12 cellulose synthase catalytic subunit transcripts, in early veraison, meanwhile veraison onwards showed more down regulated as compared to up regulated ones.3 proline-rich

29、cell wall protein related transcripts were detected throughout all the developmental stages, and in young berries the one transcript was up regulated and 2 transcripts in later stagse were up regulated.In comparison of d

30、etected reads between leaf and fruit development, leaf showed a higher number of reads.And we can also conclude from these results that the overall data generated during the analysis of both samples, that leaf showed a h

31、igher level of gene expression, however lower expression in the later stages of growth.We have reported a detailed description of the expression profiles of 2.7 billion RNA transcripts in leaf and 2.8 billion in fruit sa

32、mples to the NCBI RefSeq F.vinifera collection, andshown that this is an accurate reference for transcript abundance measurements.The biosynthetic pathways and molecular function, cellular component and biological proces

33、s, the number of genes were higher in fruit as compared to leaf.Here we observed the genes which are related to the same network but expressed differently.Gene functions are developmental stage and organ specific and reg

34、ulate networks accordingly; the growth of the plant organs is related to two main mechanisms, i.e.local mechanism and Global mechanism.Both mediums are interlinked in all parts of plants and support each other at certai