Difference between revisions of "Team:SCAU-China/Entrepreneurship"

The parts we submit are listed at the table below.

Part name	Description	Length
BBa_K1959000	PSY cds codon optimized for O.sativa.	1193 bp
BBa_K1959001	CtrⅠ cds codon optimized for O.sativa.	1650 bp
BBa_K1959002	BHY cds codon optimized for O.sativa.	1053 bp
BBa_K1959003	BKT cds codon optimized for O.sativa.	1158 bp

(1) K195900

This part is the coding sequence of phytoene synthase (PSY) from Zea mays. Codon optimized for expression in Orazy sativa. PSY is part of beta-carotene biosynthesis pathway and it catalyzes the conversion of gerarylgeranyl diphosphate to phytoene.

Reaction catalyzed by PSY

2 geranylgeranyl diphosphate → 15-cis-phytoene + diphosphate.

(2) K1959001

This part is the phytoene desaturase (Crt I) from Erwinia uredovora fused with Pea transit peptide. It is codon optimized for Orazy sativa. Bacterial phytoene desaturase catalyzes conversion of phytoene to lyscoene.

Reaction catalyzed by Crt I

15-cis-phytoene + 4 Acceptors → all-trans-lycopene + 4 Reduced acceptors

（3）K1959002

This part is the beta-carotene hydroxylase (BHY) coding sequence from Haematococcus pluvialis. The Pea transit peptide is functionally fused with BHY in order to help BHY polypeptide folding correctly. BHY sequence is codon optimized for expression in Orazy sativa. The BHY is part of astaxanthin biosynthesis pathway and it catalyzes conversion of beta-carotene to zeaxanthin.

Reaction catalyzed by BHY

Beta-carotene + 2NADH + Oxygen → Zeaxanthin + 2NAD+ + 2H2O

(4)K1959003

This part containg Pea transit peptide and coding sequence of beta-carotene ketolase (BKT). BKT catalyzes zeaxanthin to astaxanthin. Codon optimization has been made for expression in Orazy sativa. Reaction catalyzed by BKT

Zeaxanthin + 2 Oxygen → Astaxanthin + 2H2O

Transcription levels of the four transgenes (PSY, Crt I, BHY, BKT) are analyzed by performing semi-quantitative RT-PCR, Actin is served as internal control. Total RNA is extracted from astaxanthin-containing seeds and amplified using specific primers. Expected band representing the correspond genes can be detected in transformants while no band is observed in wild-type.

The RT-PCR result demonstrates that all of four transgenes is capable to transcript in rice endosperm. To a certain extent, the result also indicates that the astaxanthin biosynthesis pathway is worked in the rice endosperm.

Fig.N RT-PCR analysis of expression of CrtI, PSY, BKY,BHT genes intransformants, Actin serves as internal control. CK+, positive control (PCR product amplified from plasmid pYLTAC380MF-BBPC using specific primers ).

WT, negetive control (RT-PCR product of HG1 total RNA).

Polished transgenic seeds present in a orange color, indicating certain astaxanthin content. Wild-type rice without astaxanthin accumulation remains colorless. Inside situation of the endosperm is shown at the cross-section photo. The entire endosperm is stained in orange, representing the homogeneous distribution of astaxanthin.

Fig.n The top and cross-sectional view of phenotype of Huguang1 (O.sativa.spp.indica) wild-type and transgenic rice.

The T1 generation seeds of wild-type and transgenic lines are subjected to astaxanthin extraction and high performance liquid chromatography (HPLC) to analyze the pigment composition and astaxanthin content. Astaxanthin is indentified on the basis of retention times relative to standard compound.

Content is determined by integrating peak areas and converted to concentration. According to the retention time of standard astaxanthin compound, astaxanthin production in rice endosperm can be confirmed.

The HPLC also result shown that astaxanthin is the predominant carotenoid, while some new compounds are synthesized in conjunction with astaxanthin production. These compounds may be the intermediate products during astaxanthin biosynthesis. Further investigations are needed to identify these new compounds. Astaxanthin content in 1g of polished rice in both transgenic and wild-type rice are shown at Table.2 .

Table.2 Astaxanthin content in wild-type and transgenic rice

HG-WT	HG-380MF-BBPC
0ug/g	7.91 ug/g

The T2 generation (the third generation ) seeds were harvested at the end of September this year and we performed quantitative RT-PCR as well as HPLC analysis subsequently. Several transgenic lines were chosen for investigation.

As the results shown in the diagram, astaxanthin content varies from lines, some lines remain stable astaxanthin production while some synthesize in a low level. Supported by the quantitative RT-PCR analysis, the low content of astaxanthin in the endosperm of transgenic lines are associated with poor transcriptional activity of one of genes (BHY, which no obvious transcriptional activity is detected in four lines). The investigation demonstrates that astaxanthin biosynthesis accomplishes only under the well cooperation of the four genes, repression occurred on just one gene will badly diminish the astaxanthin content. Reasons why BHY gene is inactive in some T2 lines remain unknown and further research is needed to figure out the origins of this phenomenon.

Fig N Relative expression levels of astaxanthin biosynthetic genes and astaxanthin content in seeds of wild-type and T2 generation of transformants.

The levels of expression are normalized to actin transcript level which is set as 1.

The data represent average values from the analysis of three different plants.

Error bars indicate ± SEM. dwt, dry weight.