Cloning Cdna of Melilotis Alba

Abstract

Melilotus alba is a legume plant of the Fabaceae family. It is an invasive species, likely a descendant of a failed farming project in Alaska in 1913. The object of this experiment is to sequence the chloroplast DNA of M. alba, so as to explore its origin through uniparental cytoplasmic inheritance and mapping of its genome. The plant cell had its cpDNA removed and fragmented via EcoR1. Next, it was inserted into pUC18, a plasmid vector, and cloned in E. coli. The resulting clones were digested and ran through electrophoresis to identify contaminant DNA. Potentially pure cpDNA was selected to be sent to Yale for sequencing. Sequencing could not conclude direct relationship between the DNA and known DNA sequences of Melilotus alba. However, the DNA sequence specifically showed a strong match to the Trifolium subterraneum chloroplast genome, which is a clover plant from Europe. This suggests that M. alba was at least partially cloned. Not enough DNA was successfully sequenced to confirm current day's M. alba to the plant to Alaska in 1913.

Introduction

Melilotus alba is a legume of the Fabacaea family. It is a nitrogen fixing plant that is related to clovers. The agriculture community has found uses for it as a forage crop of cover crop. It was its use as a forage crop that brought the plant to Alaska for agricultural development in 1913. However, the plant initially did not grow as well as hoped, and was selectively bred for different varieties that might survive better. Eventually the breeding effort was deserted. Nearly a century later, M. alba has made a strong resurgence and became an invasive species. What has changed in the environment and/or the plant that has allowed it thrive? Are the plants the same that were introduced in the early 1900's, or are they from a different source?

Exploration into M.alba's form of cytoplasmic inheritance will help shed light on these questions. There are three types of cytoplasmic inheritance; vegetative segregation, uniparental inheritance, and biparental inheritance. Chloroplasts utilize uniparental inheritance, that is inheritance bias towards one parent for the donation of genetic material. Generally this is a maternal bias. This inhibits DNA recombination in non-nuclear DNA, making DNA changes generally a product of mutation. Analysis of this DNA's genetic variation, especially against other M. alba DNA, makes it possible to track the plants evolutionary and geographic history.

It will be vital to understand the origin and properties of chloroplast DNA (cpDNA) in M. alba. Chloroplasts are believed to be symbiotic organelles that were once green prokaryotes consumed by phagocytosis by proto-eukaryotes. Their DNA is estimated to be between 115 thousand to 160 thousand kbps in length, and encoded for up to 200 genes. CpDNA is circular in form, unlike nuclear DNA, which is linear.

The object of this experiment is to sequence a significant segment of M. alba's genome. E. coli will be used to clone the cpDNA and pUC18 plasmid vectors wil be used for their EcoR1 restriction site and for their ease of purifying out of the E.coli DNA.

Materials and Methods

The experiment was started with homogenizing the M. alba plant tissue. Using plants grown by Dr. Ben Harrison, the plants were cut at the soil line with razor blades and washed in a sink to remove contaminants. After being washed they were weighed for mass. The plant tissue was then chopped up with a knife and added to an ice-cold high-salt homogenizing buffer in a cold beaker at a ratio of 3mL of buffer per gram of tissue. The resulting mixture was run through a blender for bursts of 2-3 seconds until homogenous. The homogenous mixture is poured through a loose cheese cloth filter over a funnel, into another beaker on ice. This filtration process was repeated with a finer filter to catch the smaller unblended chunks that remained, and then the homogenate was poured into 50mL centrifugation tubes through a Miracloth filter. The tubes were centrifuged at 3000 x g for 10 minutes at 4˚C in a HS4 swinging rotor. Post centrifugation, the supernatant was removed. The pellet was re-suspended with the cold, high salt buffer, and centrifuged again for 5 minutes at 3000 x g. Once again, the supernatant was poured out. The pellet was then suspended in a 1 mL "TE" buffer, which inhibited the DNases, and then microfuged at 4000 x g for 2 minutes.

To break up the proteins and RNA, a lysis solution was added to the TE buffer solution at a ratio of 1:1. To mechanically disrupt the chloroplasts, the solution was plunged exactly 20 times. -mercuptoethanol and a DNA extraction buffer were added to the solution, and it was placed in incubation for 10 minutes at 65˚C. Cold potassium acetate was then added at a volume of one third the chloroplast solution. The solution was split into two tubes and centrifuged at 12,000 x g for 15 minutes at 4˚C. 1mL of the supernatant was removed and placed in a 2.0mL tube. 1mL of isopropanol was then added to the tube, the tube was inverted 6 times for proper mixture, and then allowed to incubate for 10 minutes at room temperature. The tube was then microfuged for 10 minutes. When done, the supernatant was discarded and the pellet was exposed to a fume hood for about 5 minutes. When the pellet had adequate time to dry without letting all liquid completely evaporate, 500uL of a 70% ethanol solution was added to the pellet and vortexed the tube. Then the tube was centrifuged at max speed for 5 minutes. The supernatant was aspirated out and the pellet was left open for a week, so as to let all the alcohol evaporate.

To re-dissolve the pellet, it was mixed with 20µL of Tris Buffer, flicked for mixture, and incubated in a 60˚C hot bath for one hour. According to the lab protocol, digestion of pUC18 and cpDNA was completed and then incubated at 37˚C for 60 minutes. The 2µL of cpDNA and pUC18 were diluted with 98µL of diH2O, creating a 1:50 dilution for the spectrophotometer. After approximately an hour, the digestion was complete and the EcoR1 and SAP enzymes were deactivated by a 20 minute, 65˚C hot block. Ligation of the digests was completed according to the lab manual and they were stored at 4˚C until the next lab session.

10 µL of the ligase reactions, pUC18+EcoR1, the uncut pUC18, cpDNA, and a ladder were ran through electrophoresis. Loading dye was added pre-electrophoresis to prevent the DNA from floating upwards. The electrophoresis process took approximately 45 minutes at 119 volts. The gel was then photographed in a UV light box and discarded.

E. coli was transformed when 1µl of each ligation was added to 15µLs of E.coli. To select for the desired vectors, an agar growth plate was exposed to each solution. The plate contained